Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.

Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

First report of blaOXA-181-carrying IncX3 plasmids in multidrug-resistant Enterobacter hormaechei and Serratia nevei recovered from canine and feline opportunistic infections.

Whole-genome sequence analysis of six Enterobacter hormaechei and two Serratia nevei strains, using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing, revealed the presence of the epidemic blaOXA-181-carrying IncX3 plasmids co-harboring qnrS1 and ∆ere(A) genes, as well as multiple multidrug resistance (MDR) plasmids disseminating in all strains, originated from dogs and cats in Thailand. The subspecies and sequence types (ST) of the E. hormaechei strains recovered from canine and feline opportunistic infections included E. hormaechei subsp. xiangfangensis ST171 (n = 3), ST121 (n = 1), and ST182 (n = 1), as well as E. hormaechei subsp. steigerwaltii ST65 (n = 1). Five of the six E. hormaechei strains harbored an identical 51,479-bp blaOXA-181-carrying IncX3 plasmid. However, the blaOXA-181 plasmid (pCUVET22-969.1) of the E. hormaechei strain CUVET22-969 presented a variation due to the insertion of ISKpn74 and ISSbo1 into the virB region. Additionally, the blaOXA-181 plasmids of S. nevei strains were nearly identical to the others at the nucleotide level, with ISEcl1 inserted upstream of the qnrS1 gene. The E. hormaechei and S. nevei lineages from canine and feline origins might acquire the epidemic blaOXA-181-carrying IncX3 and MDR plasmids, which are shared among Enterobacterales, contributing to the development of resistance. These findings suggest the spillover of significant OXA-181-encoding plasmids to these bacteria, causing severe opportunistic infections in dogs and cats in Thailand. Surveillance and effective hygienic practice, especially in hospitalized animals and veterinary hospitals, should be urgently implemented to prevent the spread of these plasmids in healthcare settings and communities. IMPORTANCE: blaOXA-181 is a significant carbapenemase-encoding gene, usually associated with an epidemic IncX3 plasmid found in Enterobacterales worldwide. In this article, we revealed six carbapenemase-producing (CP) Enterobacter hormaechei and two CP Serratia nevei strains harboring blaOXA-181-carrying IncX3 and multidrug resistance plasmids recovered from dogs and cats in Thailand. The carriage of these plasmids can promote extensively drug-resistant properties, limiting antimicrobial treatment options in veterinary medicine. Since E. hormaechei and S. nevei harboring blaOXA-181-carrying IncX3 plasmids have not been previously reported in dogs and cats, our findings provide the first evidence of dissemination of the epidemic plasmids in these bacterial species isolated from animal origins. Pets in communities can serve as reservoirs of significant antimicrobial resistance determinants. This situation places a burden on antimicrobial treatment in small animal practice and poses a public health threat.

A novel perspective on eugenol as a natural anti-quorum sensing molecule against Serratia sp.

Serratia marcescens is commonly noted to be an opportunistic pathogen and is often associated with nosocomial infections. In addition to its high antibiotic resistance, it exhibits a wide range of virulence factors that confer pathogenicity. Targeting quorum sensing (QS) presents a potential therapeutic strategy for treating bacterial infections caused by S. marcescens, as it regulates the expression of various virulence factors. Inhibiting QS can effectively neutralize S. marcescens' bacterial virulence without exerting stress on bacterial growth, facilitating bacterial eradication by the immune system. In this study, the antibacterial and anti-virulence properties of eugenol against Serratia sp. were investigated. Eugenol exhibited inhibitory effects on the growth of Serratia, with a minimal inhibitory concentration (MIC) value of 16.15 mM. At sub-inhibitory concentrations, eugenol also demonstrated antiadhesive and eradication activities by inhibiting biofilm formation. Furthermore, it reduced prodigiosin production and completely inhibited protease production. Additionally, eugenol effectively decreased swimming and swarming motilities in Serratia sp. This study demonstrated through molecular modeling, docking and molecular dynamic that eugenol inhibited biofilm formation and virulence factor production in Serratia by binding to the SmaR receptor and blocking the formation of the HSL-SmaR complex. The binding of eugenol to SmaR modulates biofilm formation and virulence factor production by Serratia sp. These findings highlight the potential of eugenol as a promising agent to combat S. marcescens infections by targeting its virulence factors through quorum sensing inhibition.

A long-term survey of Serratia spp. bloodstream infections revealed an increase of antimicrobial resistance involving adult population.

Serratia spp. is a well-recognized pathogen in neonates; however, limited data are available in adults. We studied microbiological and clinical characteristics of Serratia spp. causing bloodstream infections (BSI) in our institution (January 2005-July 2020). Overall, 141 BSI episodes affecting 139 patients were identified and medical records reviewed. Antimicrobial susceptibility was recovered from our informatics system and 118 isolates from 116 patients were available for further microbiological studies. Whole genome sequencing (WGS) was completed in 107 isolates. Incidence of Serratia BSI was 0.3/1000 overall admissions (range 0.12-0.60), with maximum prevalence (27 episodes, 19.1%) during 2017-2018. Relevant patients' clinical characteristics were 71.9% ≥60 years (n = 100), with high comorbidity rates (49%, ≥2), 23 (74.2%) of them died within 1 month of the BSI episode. WGS identified all isolates as Serratia marcescens when Kraken bioinformatics taxonomic tool was used despite some which were identified as Serratia nematodiphila (32/118) or Serratia ureilytica (5/118) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nevertheless, when using MASH distance, Serratia nevei (63/107), S. ureilytica (38/107), and S. marcescens (6/107) were assigned. Carbapenemase (blaVIM-1) and extended-spectrum ß-lactases (ESBL) (blaSHV-12) genes were found in seven and three isolates, respectively, one of them expressing both genes. The worldwide-disseminated IncL/M scaffold plasmid was identified in six VIM producers. Four genotypes were established based on their virulence factors and resistome. Serratia spp. emerged as a relevant nosocomial pathogen causing BSI in elderly patients in our hospital, particularly in recent years with a remarkable increase in antibiotic resistance. ESBL and carbapenemases production related to plasmid dissemination are particularly noteworthy.IMPORTANCESerratia spp. is the third most frequent pathogen involved in outbreaks at neonatal facilities and is primarily associated with bacteremia episodes. In this study, we characterized all causing bloodstream infection (BSI) in patients admitted to our hospital during a 16-year period (2005-2020). Despite having no neonatal intensive care unit in our hospital, this study revealed that Serratia spp. is a relevant pathogen causing BSI in elderly patients with high comorbidity rates. A significant increase of antimicrobial resistance was detected over time, particularly in 2020 and coinciding with the coronavirus disease (COVID-19) pandemic and nosocomial spread of multidrug-resistant Serratia spp. isolates. extended-spectrum ß-lactases and carbapenemases genes associated with plasmid dissemination, typically detected in other Enterobacterales species, were also identified, reinforcing the role of Serratia spp. in the antimicrobial resistance landscape. Additionally, this work highlights the need to reclassify the species of Serratia, since discrepancies were observed in the identification when using different tools.

CRISPR-Cas immunity is repressed by the LysR-type transcriptional regulator PigU.

Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.

High red/far-red ratio promotes root colonization of Serratia plymuthica A21-4 in tomato by root exudates-stimulated chemotaxis and biofilm formation.

Effective colonization on plant roots is a prerequisite for plant growth promoting rhizobacterias (PGPR) to exert beneficial activities. Light is essential for plant growth, development and stress response. However, how light modulates root colonization of PGPR remains unclear. Here, we found that high red/far red (R/FR) light promoted and low R/FR light inhibited the colonization and growth enhancement of Serratia plymuthica A21-4 (S. plymuthica A21-4) on tomato, respectively. Non-targeted metabolomic analysis of root exudates collected from different R/FR ratio treated tomato seedlings with or without S. plymuthica A21-4 inoculation by UPLC-MS/MS showed that 64 primary metabolites in high R/FR light-grown plants significantly increased compared with those determined for low R/FR light-grown plants. Among them, 7 amino acids, 1 organic acid and 1 sugar obviously induced the chemotaxis and biofilm formation of S. plymuthica A21-4 compared to the control. Furthermore, exogenous addition of five artificial root exudate compontents (leucine, methionine, glutamine, 6-aminocaproic acid and melezitose) regained and further increased the colonization ability and growth promoting ability of S. plymuthica A21-4 on tomato under low R/FR light and high R/FR light, respectively, indicating their involvement in high R/FR light-regulated the interaction of tomato root and S. plymuthica A21-4. Taken together, our results, for the first time, clearly demonstrate that high R/FR light-induced root exudates play a key role in chemotaxis, biofilm formation and root colonization of S. plymuthica A21-4. This study can help promote the combined application of light supplementation and PGPR to facilitate crop growth and health in green agricultural production.

Potential use of two Serratia strains for cadmium remediation based on microbiologically induced carbonate precipitation and their cadmium resistance.

Cadmium (Cd) presence and bioavailability in soils is a serious concern for cocoa producers. Cocoa plants can bioaccumulate Cd that can reach humans through the food chain, thus posing a threat to human health, as Cd is a highly toxic metal. Currently, microbiologically induced carbonate precipitation (MICP) by the ureolytic path has been proposed as an effective technique for Cd remediation. In this work, the Cd remediation potential and Cd resistance of two ureolytic bacteria, Serratia sp. strains 4.1a and 5b, were evaluated. The growth of both Serratia strains was inhibited at 4 mM Cd(II) in the culture medium, which is far higher than the Cd content that can be found in the soils targeted for remediation. Regarding removal efficiency, for an initial concentration of 0.15 mM Cd(II) in liquid medium, the maximum removal percentages for Serratia sp. 4.1.a and 5b were 99.3% and 99.57%, respectively. Their precipitates produced during Cd removal were identified as calcite by X-ray diffraction. Energy dispersive X-ray spectroscopy analysis showed that a portion of Cd was immobilized in this matrix. Finally, the presence of a partial gene from the czc operon, involved in Cd resistance, was observed in Serratia sp. 5b. The expression of this gene was found to be unaffected by the presence of Cd(II), and upregulated in the presence of urea. This work is one of the few to report the use of bacterial strains of the Serratia genus for Cd remediation by MICP, and apparently the first one to report differential expression of a Cd resistance gene due to the presence of urea.

Dynamic Gut Microbiota of Apolygus lucorum Across Different Life Stages Reveals Potential Pathogenic Bacteria for Facilitating the Pest Management.

Insect's gut microbiota has diverse effects on their fitness, and a comprehensive understanding of gut microbiota functions requires analyzing its diversity. Apolygus lucorum is a highly destructive pest that threatens many economically important crops in China. This study investigated the gut microbiota of A. lucorum across its life cycle using both culture-dependent and culture-independent methods. A total of 87 gut bacterial isolates were identified, belonging to 4 phyla, 27 families, and 45 genera, while Miseq sequencing detected 91 amplicon sequence variants (ASVs) assigned to 5 phyla, 28 families, and 39 genera. Proteobacteria and Firmicutes were the predominant phyla, with Staphylococcus and Serratia as the major genera. There were significant differences in the relative abundance of these genera between the nymph and adult stages. Staphylococcus was significantly more abundant in nymphs than it in adults, while Serratia was significantly more abundant in sexually mature adults than in other developmental stages. Notably, Serratia is a common opportunistic pathogen in many insects. Injecting the gut-dominant isolate Serratia marcescens verified its high pathogenicity. Additionally, immune indicators of the bug at different developmental stages supported the hypothesis that Serratia is a pathogen of A. lucorum. This study provides a foundation for understanding the role of gut bacteria in the life history of A. lucorum and developing new pest control strategies based on microbes.

Host-Cell-Dependent Roles of E-Cadherin in Serratia Invasion.

Bacteria use cell surface proteins to mediate host-pathogen interactions. Proteins responsible for cell adhesion, including E-cadherin, serve as receptors for entry into the host cell. We have previously shown that an increase in eukaryotic cell sensitivity to Serratia grimesii correlates with an increase in E-cadherin expression. On the other hand, Serratia proteamaculans invasion involves the EGFR, which can interact with E-cadherin on the surface of host cells. Therefore, we investigated the role of E-cadherin in Serratia invasion into M-HeLa and Caco-2 cells. Bacterial infection increased E-cadherin expression in both cell lines. Moreover, E-cadherin was detected in the Caco-2 cells in a full-length form and in the M-HeLa cells in only a truncated form in response to incubation with bacteria. Transfection with siRNA targeting E-cadherin inhibited S. proteamaculans invasion only into the Caco-2 cells. Thus, only full-length E-cadherin is involved in S. proteamaculans invasion. On the other hand, transfection with siRNA targeting E-cadherin inhibited S. grimesii invasion into both cell lines. Thus, not only may full-length E-cadherin but also truncated E-cadherin be involved in S. grimesii invasion. Truncated E-cadherin can be formed as a result of cleavage by bacterial proteases or the Ca2+-activated cellular protease ADAM10. The rate of Ca2+ accumulation in the host cells depends on the number of bacteria per cell upon infection. During incubation, Ca2+ accumulates only when more than 500 S. grimesii bacteria are infected per eukaryotic cell, and only under these conditions does the ADAM10 inhibitor reduce the sensitivity of the cells to bacteria. An EGFR inhibitor has the same quantitative effect on S. grimesii invasion. Apparently, as a result of infection with S. grimesii, Ca2+ accumulates in the host cells and may activate the ADAM10 sheddase, which can promote invasion by cleaving E-cadherin and, as a result, triggering EGFR signaling. Thus, the invasion of S. proteamaculans can only be promoted by full-length E-cadherin, and S. grimesii invasion can be promoted by both full-length and truncated E-cadherin.

Isolation and Characterization of a Serratia rubidaea from a Shallow Water Hydrothermal Vent.

Microbial life present in the marine environment has to be able to adapt to rapidly changing and often extreme conditions. This makes these organisms a putative source of commercially interesting compounds since adaptation provides different biochemical routes from those found in their terrestrial counterparts. In this work, the goal was the identification of a marine bacterium isolated from a sample taken at a shallow water hydrothermal vent and of its red product. Genomic, lipidomic, and biochemical approaches were used simultaneously, and the bacterium was identified as Serratia rubidaea. A high-throughput screening strategy was used to assess the best physico-chemical conditions permitting both cell growth and production of the red product. The fatty acid composition of the microbial cells was studied to assess adaptation at the lipid level under stressful conditions, whilst several state-of-the-art techniques, such as DSC, FTIR, NMR, and Ultra-High Resolution Qq-Time-of-Flight mass spectrometry, were used to characterize the structure of the pigment. We hypothesize that the pigment, which could be produced by the cells up to 62 °C, is prodigiosin linked to an aliphatic compound that acts as an anchor to keep it close to the cells in the marine environment.

The Promoting Mechanism of the Sterile Fermentation Filtrate of Serratia odorifera on Hypsizygus marmoreus by Means of Metabolomics Analysis.

Hypsizygus marmoreus has become one of the most popular edible mushrooms due to its high nutritional and economic value. Previous researchers found that Serratia odorifera could promote the growth of H. marmoreus by producing and secreting some of its inducers. However, the specific mechanism of action was still unclear. In this study, we found that the exogenous addition of sterile fermentation filtrate (HZSO-1), quorum sensing (QS) signaling molecules, 3-oxo-C6-HSL, cyclo(Pro-Leu), and cyclo(Tyr-Leu) could significantly promote the growth of H. marmoreus, increase the number of clamp junctions, and the diameter of mycelium (p < 0.05). In addition, non-targeted metabolomic analysis revealed that 706 metabolites were detected in the treated group. Of these, 307 metabolites were significantly different (p < 0.05). Compared with the control, 54 and 86 metabolites were significantly increased and decreased in the HZSO-1 group, respectively (p < 0.05). We speculate that the sterile fermentation filtrate of S. odorifera could mediate the carbohydrate and amino acid metabolism of H. marmoreus by influencing the pentose phosphate pathway (PPP) to increase the energy supply for the growth and development of the mycelium. The above results will further reveal the growth-promoting mechanism of S. odorifera on H. marmoreus.

In silico exploration of Serratia sp. BRL41 genome for detecting prodigiosin Biosynthetic Gene Cluster (BGC) and in vitro antimicrobial activity assessment of secreted prodigiosin.

The raising concern of drug resistance, having substantial impacts on public health, has instigated the search of new natural compounds with substantial medicinal activity. In order to find out a natural solution, the current study has utilized prodigiosin, a linear tripyrrole red pigment, as an active ingredient to control bacterial proliferation and prevent cellular oxidation caused by ROS (Reactive Oxygen Species). A prodigiosin-producing bacterium BRL41 was isolated from the ancient Barhind soil of BCSIR Rajshahi Laboratories, Bangladesh, and its morphological and biochemical characteristics were investigated. Whole genome sequencing data of the isolate revealed its identity as Serratia sp. and conferred the presence of prodigiosin gene cluster in the bacterial genome. "Prodigiosin NRPS", among the 10 analyzed gene clusters, showed 100% similarity with query sequences where pigC, pigH, pigI, and pigJ were identified as fundamental genes for prodigiosin biosynthesis. Some other prominent clusters for synthesis of ririwpeptides, yersinopine, trichrysobactin were also found in the chromosome of BRL41, whilst the rest displayed less similarity with query sequences. Except some first-generation beta-lactam resistance genes, no virulence and resistance genes were found in the genome of BRL41. Structural illumination of the extracted red pigment by spectrophotometric scanning, Thin-Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FTIR), and change of color at different pH solutions verified the identity of the isolated compound as prodigiosin. Serratia sp. BRL41 attained its maximum productivity 564.74 units/cell at temperature 30˚C and pH 7.5 in two-fold diluted nutrient broth medium. The compound exhibited promising antibacterial activity against Gram-positive and Gram-negative bacteria with MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values ranged from 3.9 to15.62 µg/mL and 7.81 to 31.25 µg/mL respectively. At concentration 500 µg/mL, except in Salmonella enterica ATCC-10708, prodigiosin significantly diminished biofilm formed by Listeria monocytogens ATCC-3193, Pseudomonas aeruginosa ATCC-9027, Escherichia coli (environmental isolate), Staphylococcus aureus (environmental isolate). Cellular glutathione level (GSH) was elevated upon application of 250 and 500 µg/mL pigment where 125 µg/mL failed to show any free radical scavenging activity. Additionally, release of cellular components in growth media of both Gram-positive and Gram-negative bacteria were facilitated by the extract that might be associated with cell membrane destabilization. Therefore, the overall findings of antimicrobial, antibiofilm and antioxidant activities suggest that in time to come prodigiosin might be a potential natural source to treat various diseases and infections.

Genome-driven discovery of new serrawettin W2 analogues from Serratia fonticola DSM 4576.

By expressing a multimodular NRPS gene sefA from Serratia fonticola DSM 4576 in E. coli, four new serrawettin W2 analogues, namely sefopeptides A-D (1-4), were isolated and structurally characterized and their biosynthesis was proposed. A bioactivity assay showed that sefopeptide C (3) exhibits moderate cytotoxic activity against acute promyelocytic leukemia NB4 cells.

Isolation and identification of specific lytic bacteriophages as a biocontrol agent against Serratia odorifera PBA-IAUF-1 and Kluyvera intermedia PBA-IAUF-6 causing bacterial canker in the grape and Siberian pear.

Bacterial canker, a prevalent disease among fruit trees, is a significant concern. The use of phage therapy is presently seen as a dependable biological strategy to control bacterial diseases in fruits. The objective of this research was to use various biochemical and molecular techniques to determine the types of bacteria responsible for causing cankers in various fruits. Additionally, their ability to cause disease in the fruit tissues was assessed, the specific bacteriophages targeting these bacteria were isolated and identified. The bacteria were separated from different parts of the infected fruits like grapes and Siberian pears. The selection of fruit tissues showing signs of canker disease was performed, and the validation of the isolates' pathogenicity was confirmed following Koch's principles. Subsequently, in order to establish a conclusive identification of the bacterial species, molecular identification was conducted through the sequencing of a specific fragment within the 16S rRNA following amplification by PCR by using universal primers, RW01 and DG74. Isolation and titration of phages specific to fruit spoilage bacteria was done by spot and double-layer agar method, and the growth curve of the isolated bacteriophage was drawn. The phages were detected by transmission electron microscopy (TEM). The results of the study proved the presence of canker causing agents, Kluyvera intermedia PBA-IAUF-6 with the code Sh6 in the Siberian pears, and Serratia odorifera PBA-IAUF-1 with the code Rz3 in the grape fruits, which were deposited in GenBank with the accession numbers of KU878579 and KU168605, respectively. Isolation of the specific bacteriophages to the S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 bacterial strains were done from the effluent of South Isfahan wastewater treatment plant and Caspian Sea water, respectively. The titer of the specific phage to S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 was detected in the samples as 2.2 × 10-5 and 5 × 10-11 PFU/ml, respectively. An electron micrograph of a bacteriophage that targets two different bacterial strains revealed phages with a geometrically shaped head and a flexible tail, which resembled viruses from the Siphoviridae family.

Prevalence of carbapenem-resistant and extended-spectrum beta-lactamase-producing Enterobacteriaceae in a teaching hospital in Ghana.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) and Extended-spectrum beta-lactamase (ESBL) production among Gram-negative Enterobacteriaceae is an increasing global challenge due to the high morbidity and mortality associated with their infections, especially in developing countries where there are little antibiotic treatment options. Despite these challenges, few studies in Ghana have described the burden of CRE. Therefore, this study aimed to determine the prevalence of carbapenem-resistant Enterobacteriaceae isolated from patients at the Cape Coast Teaching Hospital (CCTH) in the Central region of Ghana. METHODOLOGY/PRINCIPAL FINDINGS: Enterobacteriaceae isolates were collected from April to July 2019 at the bacteriology unit of CCTH using a consecutive sampling method. Isolates were identified by standard microbiological techniques and confirmed using API 20E. Kirby Bauer disc diffusion method was used to determine the antibiogram of isolates. Isolates were also subjected to ESBL testing using the single-disc combination method. Carbapenem-resistant isolates were identified by the Kirby Bauer disc diffusion method and then examined genotypically for the presence of blaKPC-1, blaIMP-1, blaVIM-1, blaNDM-1, and blaOXA-48 genes via polymerase chain reaction (PCR). Of the 230 isolates comprising E. coli (40.9%), Citrobacter spp. (32.6%), K. pneumoniae (9.1%), P. mirabilis (6.1%), P. vulgaris (5.2%), Enterobacter spp (3.5%)., K. oxytoca (2.2%), and Serratia marcenses (0.4%). Most isolates were from urine 162(70.4%) and wound samples. The isolates showed high resistance to ampicillin 171 (74.3%) and cefuroxime 134(58.3%). The prevalence of MDR was 35.2% (81), with E. coli 40(42.6%) being the majority that exhibited MDR. Of the 230 isolates, 113(49.1%) were ESBL producers, with E. coli 54(57.5%) accounting for the majority, while Serratia marcenses was the least. Of the 13 (5.7%) CRE isolates that showed resistance towards carbapenem in the disc diffusion method, 11 showed the presence of the blaNDM-1 gene, while all isolates showed the presence of the blaOXA-48 gene. CONCLUSION: The prevalence of carbapenem resistance and ESBL-producing Enterobacteriaceae pathogens among patients at the Cape Coast Teaching Hospital is high and alarming. Therefore, it is imperative to consider effective infection prevention and control measures should be implemented at the hospital to prevent the rapid spread of these dangerous organisms.

Rhs NADase effectors and their immunity proteins are exchangeable mediators of inter-bacterial competition in Serratia.

Many bacterial species use Type VI secretion systems (T6SSs) to deliver anti-bacterial effector proteins into neighbouring bacterial cells, representing an important mechanism of inter-bacterial competition. Specific immunity proteins protect bacteria from the toxic action of their own effectors, whilst orphan immunity proteins without a cognate effector may provide protection against incoming effectors from non-self competitors. T6SS-dependent Rhs effectors contain a variable C-terminal toxin domain (CT), with the cognate immunity protein encoded immediately downstream of the effector. Here, we demonstrate that Rhs1 effectors from two strains of Serratia marcescens, the model strain Db10 and clinical isolate SJC1036, possess distinct CTs which both display NAD(P)+ glycohydrolase activity but belong to different subgroups of NADase from each other and other T6SS-associated NADases. Comparative structural analysis identifies conserved functions required for NADase activity and reveals that unrelated NADase immunity proteins utilise a common mechanism of effector inhibition. By replicating a natural recombination event, we show successful functional exchange of CTs and demonstrate that Db10 encodes an orphan immunity protein which provides protection against T6SS-delivered SJC1036 NADase. Our findings highlight the flexible use of Rhs effectors and orphan immunity proteins during inter-strain competition and the repeated adoption of NADase toxins as weapons against bacterial cells.

ShlA toxin of Serratia induces P2Y2- and α5ß1-dependent autophagy and bacterial clearance from host cells.

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.

Phages on filaments: A genetic screen elucidates the complex interactions between Salmonella enterica flagellin and bacteriophage Chi.

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such as Salmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to the S. enterica flagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certain Salmonella serotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of the Salmonella flagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility in S. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host genera Salmonella, Escherichia, or Serratia.

Serratia endocarditis: antimicrobial management strategies and clinical outcomes.

OBJECTIVES: The incidence of Serratia endocarditis is increasing, yet optimal treatment has not been defined. Our objective was to investigate the outcomes of patients with Serratia endocarditis by treatment strategy. METHODS: We reviewed adult patients with definitive Serratia endocarditis at two independent health systems between July 2001 and April 2023. Combination therapy was defined as receipt of ≥2 in vitro active agents for ≥72 h. RESULTS: Seventy-five patients were included; 64% (48/75) were male and 85% (64/75) were people who inject drugs. Compared with monotherapy, receipt of combination therapy was associated with lower rates of microbiological failure (0% versus 15%, P = 0.026) and 90 day all-cause mortality (11% versus 31%, P = 0.049). Antimicrobial discontinuation due to an adverse event was more common among patients receiving combination therapy compared with monotherapy (36% versus 8%, P = 0.058). CONCLUSIONS: In the largest series of Serratia endocarditis to date, combination antibiotic treatment was associated with improved outcomes. However, larger, prospective studies are warranted.