Virulence Genes Research Articles

Evolutionary and functional divergence of Sfx, a plasmid-encoded H-NS homolog, underlies the regulation of IncX plasmid conjugation.

Conjugative plasmids are widespread among prokaryotes, highlighting their evolutionary success. Conjugation systems on most natural plasmids are repressed by default. The negative regulation of F-plasmid conjugation is partially mediated by the chromosomal nucleoid-structuring protein (H-NS). Recent bioinformatic analyses have revealed that plasmid-encoded H-NS homologs are widespread and exhibit high sequence diversity. However, the functional roles of most of these homologs and the selective forces driving their phylogenetic diversification remain unclear. In this study, we characterized the functionality and evolution of Sfx, a H-NS homolog encoded by the model IncX2 plasmid R6K. We demonstrate that Sfx, but not chromosomal H-NS, can repress R6K conjugation. Notably, we find evidence of positive selection acting on the ancestral Sfx lineage. Positively selected sites are located in the dimerization, oligomerization, and DNA-binding interfaces, many of which contribute to R6K repression activity-indicating that adaptive evolution drove the functional divergence of Sfx. We additionally show that Sfx can physically interact with various chromosomally encoded proteins, including H-NS, StpA, and Hha. Hha enhances the ability of Sfx to regulate R6K conjugation, suggesting that Sfx retained functionally important interactions with chromosomal silencing proteins. Surprisingly, the loss of Sfx does not negatively affect the stability or dissemination of R6K in laboratory conditions, reflecting the complexity of selective pressures favoring conjugation repression. Overall, our study sheds light on the functional and evolutionary divergence of a plasmid-borne H-NS-like protein, highlighting how these loosely specific DNA-binding proteins evolved to specifically regulate different plasmid functions.IMPORTANCEConjugative plasmids play a crucial role in spreading antimicrobial resistance and virulence genes. Most natural conjugative plasmids conjugate only under specific conditions. Therefore, studying the molecular mechanisms underlying conjugation regulation is essential for understanding antimicrobial resistance and pathogen evolution. In this study, we characterized the conjugation regulation of the model IncX plasmid R6K. We discovered that Sfx, a H-NS homolog carried by the plasmid, represses conjugation. Molecular evolutionary analyses combined with gain-of-function experiments indicate that positive selection underlies the conjugation repression activity of Sfx. Additionally, we demonstrate that the loss of Sfx does not adversely affect R6K maintenance under laboratory conditions, suggesting additional selective forces favoring Sfx carriage. Overall, this work underscores the impact of protein diversification on plasmid biology, enhancing our understanding of how molecular evolution affects broader plasmid ecology.

Characterization and genomic analysis of Salmonella Abortusequi phage, vB_SalP_LDDK01, and its biocontrol application in donkey meat

Salmonella Abortusequi (S. Abortusequi) is the primary cause of abortions in equine animals, and can cause serious foodborne illness. Thus, effective biocontrol strategies are needed to decontaminate and control the emergence of foodborne diseases. In recent years, phages have been used as a new strategy for modulating foodborne pathogens and food safety. In this study, a new phage, vB_SalP_LDDK01, was isolated from donkey farm bedding. The data indicated that the incubation period of vB_SalP_LDDK01 was 10 min, the burst size was 378 PFU/cell, as well as a wide range of heat resistance (40-70°C) and pH stability (4-12). Furthermore, genomic analysis and electron microscopy indicated that vB_SalP_LDDK01 belongs to the Class Caudoviricetes and genus Jerseyvirus. Moreover, its genome was 42,378 bp long, encoded 57 ORFs, was double-stranded DNA with a 49.52% GC content, and lacked virulence and drug-resistant genes. In addition, how vB_SalP_LDDK01 inhibits the growth of S. Abortusequi and removes the biofilm of S. Abortusequi was assessed in a liquid broth medium, and the results showed that vB_SalP_LDDK01 inhibited the growth of S. Abortusequi for about 8 h and significantly reduced the viable bacteria abundance compared with the phage-free positive control. Further, vB_SalP_LDDK01 treated the host bacteria for 12 h and effectively destroyed the biofilm of S. Abortusequi. This study further investigated how effectively vB_SalP_LDDK01 modulates bacterial contamination in donkey meat inoculated with S. Abortusequi LCU-S-ABORT-F at 4°C and 25°C. Furthermore, after 72 h of vB_SalP_LDDK01 treatment with different multiplicity of infection (1, 0.1, 0.01, and 0.001), the bacterial contamination on the surface of donkey meat was reduced by 4.3, 3.7, 3.3, and 3.5 log10 CFU/piece at 25°C, and 4.5, 3.9, 2.8, and 2.7 log10 CFU/piece at 4°C. Whereas the phage titers at different temperatures were basically comparable to the initial titers. Overall, these results indicated that vB_SalP_LDDK01, the new phage, can serve as an effective biological agent and inhibit S. Abortusequi in donkey meat.

Vancomycin-resistant Staphylococcus aureus endangers Egyptian dairy herds.

The emergence of pandrug-resistant (PDR) and extensive drug-resistant (XDR) methicillin-resistant and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) isolates from bovine milk samples along with biofilm formation ability and harboring various virulence genes complicates the treatment of bovine mastitis and highlights the serious threat to public health. This study investigated for the first time the frequency, antimicrobial resistance profiles, biofilm-forming ability, virulence factors, spa and staphylococcal cassette chromosome mec (SCCmec) types of MRSA and VRSA isolated from clinical and subclinical bovine mastitis in Egypt. A total of 808 milk samples were collected from each quarter of 202 dairy animals, including 31 buffaloes and 171 cattle. The frequency of mastitis in the collected milk samples was 48.4% (60/124) in buffaloes and 29.2% (200/684) in cattle. A total of 65 Staphylococcus species isolates were recovered, including 27 coagulase-positive S. aureus (CoPS) isolates and 38 coagulase-negative staphylococci (CoNS). The CoNS included 27 mammaliicocci (20 Mammaliicoccus lentus and 7 M. sciuri) and 11 Non-aureus staphylococci (S.lugdunensis) isolates. All the CoPS isolates were mecA positive and resistant to 20-33 tested antimicrobials with multiple antibiotic resistance index ranging from 0.61 to 1. Three isolates were PDR, four were XDR, and 20 were multidrug resistant isolates. VRSA was detected in 85.2% of CoPS isolates with minimal inhibitory concentration (MIC) ranging from 64 to 1024µg/mL. The vanA gene was found in 60.8%, vanB in 73.9%, and both genes in 43.5% of VRSA isolates. All the CoPS isolates exhibited biofilm formation ability, with 55.6% being strong, and 44.4% moderate biofilm producers, and harbored icaA (74.1%) and icaD (74.1%) biofilm-forming genes. All S. aureus isolates harbored both beta-haemolysin (hlb) and leucotoxin (lukMF) genes, while 44.4% were positive for toxic shock syndrome toxin (tsst) gene. Enterotoxin genes sea, seb, sec, sed, and see were found in 59.3%, 40.7%, 18.5%, 33.3%, and 14.8% of isolates, respectively. Additionally, 70.4% of the isolates hadspa X-region gene, and exhibited eight different MRSA spa types (t127, t267, t037, t011, t843, t1081, t2663, and t1575), with spa t127 being the most common. Three SCCmec types (I, II and III) were identified, with SCCmec I being predominant, and were further classified into subtypes 1.1.1, 1.1.2, 1.n.1, and 4.1.1. The ability of MRSA and VRSA isolates to produce biofilms and resist antimicrobials highlights the serious threat these pathogens pose to bovine milk safety, animal welfare, and public health. Therefore, strict hygiene practices and antimicrobial surveillance are crucial to reduce the risk of MRSA and VRSA colonization and dissemination.

An Epidemiological Study on Salmonella in Tibetan Yaks from the Qinghai-Tibet Plateau Area in China.

Salmonella is an important foodborne pathogen that can cause a range of illnesses in humans; it has also been a key focus for monitoring in the field of public health, including gastroenteritis, sepsis, and arthritis, and can also cause a decline in egg production in poultry and diarrhea and abortion in livestock, leading to death in severe cases, resulting in huge economic losses. This study aimed to investigate the isolation rate, antimicrobial resistance, serotypes, and genetic diversity of Salmonella isolated from yak feces in various regions on the Qinghai-Tibet Plateau. A total of 1222 samples of yak dung were collected from major cities in the Qinghai-Tibet Plateau area, and the sensitivity of the isolated bacteria to 10 major classes of antibiotics was determined using the K-B paper disk diffusion method for drug susceptibility. Meanwhile, the serotypes of the isolated bacteria were analyzed using the plate agglutination test for serum antigens, and their carriage of drug resistance and virulence genes was determined using PCR and gel electrophoresis experiments. The isolated bacteria were also classified using MLST (Multi-Locus Sequence Typing). The overall isolation rate for Salmonella was 18.25% (223/1222), and the results of the antibiotic susceptibility tests showed that 98.65% (220/223) of the isolated bacteria were resistant to multiple antibiotics. In the 223 isolates of Salmonella, eight classes of 20 different resistance genes, 30 serotypes, and 15 different types of virulence genes were detected. The MLST analysis identified 45 distinct sequence types (STs), including five clonal complexes, of which ST34, ST11, and ST19 were the most common. These findings contribute valuable information about strain resources, genetic profiles, and typing data for Salmonella in the Qinghai-Tibet Plateau area, facilitating improved bacterial surveillance, identification, and control in yak populations. They also provide certain data supplements for animal Salmonella infections globally, filling research gaps.

Biological features and medical significance of the <i>Listeria</i> bacteria

Bacteria of the genus Listeria are widely distributed in the environment; they are isolated from soil and water ecosystems, food products, environmental objects, and circulate in vivo. L. monocytogenes are pathogenic for animals and humans. The ecological plasticity, stress resistance and tolerance of Listeria determines their ability to switch from a saprophytic to a parasitic life cycle and survive under various environmental conditions. After thawing and subsequent cultivation of Listeria on fresh nutrient medium, a pronounced populational heteromorphism is noted: formation of protoplast-type cells, L-forms and convoluted revertant cells, which requires the use PCR and ELISA for bacteria detection. It is known that non-pathogenic Listeria, as well as pathogenic microorganisms forming a biocenosis with L. monocytogenes, can serve as a reservoir of pathogenicity and resistance determinants and be transferred to pathogenic Listeria by horizontal transfer, which leads to the emergence of new, more virulent and antibiotic-resistant strains. In addition, the most important adaptation mechanism of L. monocytogenes to adverse environmental factors is their ability to form biofilms markedly enhancing survival and disinfectant resistance. The relatively high genomic similarity between L. monocytogenes and some non-pathogenic Listeria species and often their coexistence in similar ecological niches, may provide an opportunity for the transfer of resistance or virulence genes. At the same time, the transmission of pathogenicity genes from L. monocytogenes to L. innocua is also possible, which predetermines the appearance of atypical hemolytic pathogenic strains, which, given the greater environmental prevalence of the latter can pose a great danger to humans and animals. The increasing role of Listeria in the pattern of human and animal infectious pathologies, the variability of their morphological, cultivable and biochemical properties, as well as the constant modification of the surface Listeria antigens underlies a need to improve listeriosis diagnostics and requires creation of new immunobiological preparations and modern regimens for isolation and identification of various Listeria types. This review discusses current views on Listeria spp. prevalence and biological qualities, virulence and pathogenicity factors of L. monocytogenes, as well as methods for identifying different Listeria species.

Emergence and genetic characterization of KLUC-3 extended-spectrum β-lactamase-producing Escherichia coli ST95 High-Risk clone causing nosocomial infection in Japan.

KLUC β-lactamase is a minor extended-spectrum β-lactamase (ESBL) derived from chromosome-encoded cefotaximase in Kluyvera cryocrescens. This study aimed to characterize the genetic context of KLUC-3-producing Escherichia coli and blaKLUC-3-harboring plasmids and assess nosocomial transmission. In a national genomic surveillance conducted in 2019 and 2020, KLUC-3-producing E. coli strains (JBEAACH-19-0093 and JBEAACH-19-0210) were recovered from two pediatric inpatients in a Japanese hospital. Short- and long-read sequencing analyses using the HiSeq X Five and GridION were performed to determine the complete genome sequences. JBEAACH-19-0093 and JBEAACH-19-0210 belong to the B2-O1:K1:H7-ST95-fimH41 global high-risk clones and carry virulence genes related to extraintestinal pathogenic and uropathogenic E. coli. Single nucleotide polymorphism analysis showed high homology (13 SNPs) between the strains, suggesting nosocomial transmission. The blaKLUC-3 gene was flanked by ISEcp1 and Δorf477 on 100-kb IncB/O/K/Z plasmids with a complete sequence identity. Comparative analysis revealed ISEcp1-mediated transposition of blaKLUC-3 into the IncB/O/K/Z plasmid; the complete plasmid sequence was highly similar to blaCTX-M- and blaCMY-2-harboring plasmids from E. coli ST131 isolates. In GenBank database, 26 Enterobacterales harbored blaKLUC-1 to blaKLUC-7, particularly in Asia. Among them, the genomic structure of blaKLUC-orf477/Δorf477 is conserved in 23 strains. In 13 Enterobacterales, except K. cryocrescens, ISEcp1 was inserted upstream of blaKLUC and 10 strains carried blaKLUC on the plasmids. This is the first case of nosocomial transmission of KLUC-3 producers outside China. The blaKLUC-3 emergence in the virulent pandemic lineage ST95 is a public health problem highlighting the need for further investigations to prevent its potential dissemination.

Genomic Characterization of Probiotic Purple Nonsulfur Bacteria Cereibacter sphaeroides Strains S3W10 and SS15: Implications for Enhanced Shrimp Aquaculture.

Cereibacter sphaeroides strains S3W10 and SS15, isolated from shrimp ponds, exhibit potential probiotic benefits for aquaculture. In this study, the genomic features of S3W10 and SS15 were thoroughly characterized to evaluate their probiotic properties and safety for aquaculture use. The genomes of S3W10 and SS15 consist of 130 and 74 contigs, with sizes of 4.6 Mb and 4.4 Mb and GC contents of 69.2%. Average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and phylogenomic analyses confirmed that these strains belong to C. sphaeroides. Genome annotation predicted 4260 coding sequences (CDS) in S3W10 and 4086 CDS in SS15, including genes associated with stress tolerance, nutrient absorption, and antioxidant activity. Notably, genes related to vitamin B12 synthesis, digestive enzyme production, and carotenoid biosynthesis, which support shrimp health, were identified in both genomes. CAZyme analysis identified 116 and 115 carbohydrate-active enzymes in S3W10 and SS15, respectively, supporting adaptation to gastrointestinal environments and the host immune response. Pan-genome analysis across C. sphaeroides strains revealed 7918 gene clusters, highlighting the open pan-genome structure of this species and its high genetic diversity. Further bioinformatic analyses assessing mobile genetic elements, antibiotic-resistance genes, and virulence factors demonstrated the safety of both strains for aquaculture, as no plasmids or virulence genes were identified. The genomic insights in this study provide a deeper understanding of the strains' adaptability and functional potential, aligning with previous in vitro and in vivo studies and highlighting their potential for use in shrimp cultivation.

Pathogenomic Insights into Xanthomonas oryzae pv. oryzae's Resistome, Virulome, and Diversity for Improved Rice Blight Management.

Oryza sativa (rice) is a major staple food targeted for increased production to achieve food security. However, increased production is threatened by several biotic and abiotic factors, of which bacterial blight disease caused by Xanthomonas oryzae pathovar oryzae is severe. Developing effective control strategies requires an up-to-date understanding of its pathogenomics. This study analyzes the genomes of 30 X. oryzae strains collected from rice-producing regions across five continents to identify genetic elements critical for its pathogenicity and adaptability and for an intraspecific diversity assessment using advanced genomics and bioinformatics tools. Resistome analysis revealed 28 distinct types of antibiotic resistance genes (ARGs), both innate and acquired, indicating a growing threat from multidrug-resistant X. oryzae strains. Sixteen virulent genes, including type III and VI secretion systems, motility genes, and effector proteins, were identified. A unique 'MexCD-OprJ' multidrug efflux system was detected in the Tanzanian strains, conferring resistance to multiple antibiotic classes. To curb further ARG emergence, there is a need to regulate the use of antibiotics for X. oryzae control and adopt resistant rice varieties. Transposable elements were also discovered to contribute to X. oryzae pathogenicity, facilitating the horizontal transfer of virulence genes. Pangenome analysis revealed intraspecific variation among the population, with 112 unique CDS having diverse functional roles. Strains registered in the Philippines had the most unique genes. Phylogenetic analysis confirmed the divergent evolution of X. oryzae. This study's results will aid in identifying more effective management strategies and biocontrol alternatives for sustainable rice production.

Detection of CRISPR‒Cas and type I R-M systems in Klebsiella pneumoniae of human and animal origins and their relationship to antibiotic resistance and virulence.

The clustered regularly interspaced short palindromic repeats (CRISPR)‒CRISPR-associated protein (Cas) and restriction‒modification (R-M) systems are important immune systems in bacteria. Information about the distributions of these two systems in Klebsiella pneumoniae from different hosts and their mutual effect on antibiotic resistance and virulence is still limited. In this study, the whole genomes of 520 strains of K. pneumoniae from GenBank, including 325 from humans and 195 from animals, were collected for CRISPR‒Cas systems and type I R-M systems, virulence genes, antibiotic resistance genes, and multilocus sequence typing detection. The results showed that host origin had no obvious influence on the distributions of the two systems (CRISPR‒Cas systems in 29.8% and 24.1%, type I R-M systems in 9.8% and 11.8% of human-origin and animal-origin strains, respectively) in K. pneumoniae. Identical spacer sequences from different hosts demonstrated there was a risk of human-animal transmission. All virulence genes (yersiniabactin, colibactin, aerobactin, salmochelin, rmpADC, and rmpA2) detection rates were higher when only the CRISPR‒Cas systems were present but were all reduced when coexisting with type I R-M systems. However, a lower prevalence of most antibiotic-resistance genes was found when the CRISPR‒Cas systems were alone, and when type I R-M systems were coexisting, some of the antibiotic resistance gene incidence rates were even lower (aminoglycosides, clindamycins, rifampicin-associated resistance genes, sulfonamides, methotrexates, beta-lactamases, and ultrabroad-spectrum beta-lactamases), and some of them were higher instead (quinolones, macrolides, tetracyclines, and carbapenems). The synergistic and opposed effects of the two systems on virulence and antibiotic-resistance genes need further study.IMPORTANCEK. pneumoniae is an important opportunistic pathogen responsible for both human and animal infections, and the emergence of hypervirulent and multidrug-resistant K. pneumoniae has made it difficult to control this pathogen worldwide. Here, we find that CRISPR‒Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, have synergistic and opposed effects on virulence and antibiotic resistance genes in K. pneumoniae. Moreover, this study provides insights into the distributions of the two systems in K. pneumoniae from different hosts, and there is no significant difference in the prevalence of the two systems among K. pneumoniae spp. In addition, this study also characterizes the CRISPR arrays of K. pneumoniae from different hosts, suggesting that the strains sharing the same spacer sequences have the potential to spread between humans and animals.

Characterization of a carbapenemase-producing Klebsiella pneumoniae isolate of a patient in an intensive care unit in Greece: A study of resistome, virulome, and mobilome.

Klebsiella pneumoniae is a major pathogen associated with hospital-acquired infections, particularly those involving multidrug-resistant strains. Carbapenem resistance, often driven by carbapenemases such as KPC, VIM, OXA-48, and NDM, poses a significant challenge in clinical settings. This study reports on K. pneumoniae strain A165, isolated from a blood culture of a 51-year-old female patient hospitalized for respiratory distress post-SARS-CoV-2 infection. This K. pneumoniae strain exhibited resistance to several antibiotics, including carbapenems, cephalosporins, aminoglycosides, and fluoroquinolones, but remained susceptible to gentamicin, colistin, and trimethoprim-sulfamethoxazole. Next-generation sequencing was performed on Ion torrent platform, that revealed a genome size of 5,676,404 bp, including a chromosome and six plasmids. The strain was classified as sequence type 11 (ST11), a high-risk lineage associated with carbapenem resistance. The resistome of A165 included multiple β-lactamase genes, such as blaNDM-1 and blaOXA-48, as well as genes conferring resistance to other antibiotic classes. The virulome analysis identified genes involved in iron acquisition (yersiniabactin operon genes: ybtE, ybtT, irp1, irp2; aerobactin receptor: iutA), adhesion (mrkA-J, fimA-K), capsule and biofilm formation (rcsA, rcsB, ompA) and resistance to complement (traT) contributing to its pathogenic potential. The mobilome analysis revealed nine insertion sequences, including ISKpn1, ISKpn18, ISKpn43, ISKpn28, ISKpn14, ISEcp1, and IS6100. The strain also harbored six replicons: Col440II, ColRNAI, IncFIA(HI1), IncFIB(K), IncFII(K), and IncR, which are associated with the horizontal transfer of resistance and virulence genes. Comparative analysis with global isolates demonstrated the widespread dissemination of carbapenemase-producing K. pneumoniae, with notable occurrences in Europe, Asia, and the Americas. This study highlights the growing concern of multidrug-resistant K. pneumoniae in hospital settings and emphasizes the need for robust surveillance and infection control measures.

Genome-wide phenotypic profiling of transcription factors and identification of novel targets to control the virulence of Vibrio vulnificus.

For successful infection, the life-threatening pathogen Vibrio vulnificus elaborately regulates the expression of survival and virulence genes using various transcription factors (TFs). In this study, a library of the V. vulnificus mutants carrying specific signature tags in 285 TF genes was constructed and subjected to 16 phenotypic analyses. Consequently, 89 TFs affecting more than one phenotype of V. vulnificus were identified. Of these, 59 TFs affectedthe in vitro survival including growth, stress resistance, biofilm formation and motility, and 64 TFs affected the virulence of V. vulnificus. Particularly, 27 of the 64 TFs enhanced the in vitro hemolytic or cytotoxic activities, and 8 of the 27 TFs also increased the in vivo brine shrimp or murine infectivities of V. vulnificus. Among the eight TFs, HlyU, IscR, NagC, MetJ and Tet2 did not affect the growth of V. vulnificus but still regulated the expression of major exotoxin genes, including rtxA, vvhA and plpA, thereby emerging as potential drug targets for anti-virulence therapies with low selective pressure for developing resistance. Altogether, this study characterized the functions of TFs at a genome-wide scale and identified novel targets to control the virulence of V. vulnificus.

Characterization of a Novel KPC-2 Variant, KPC-228, Conferring Resistance to Ceftazidime-Avibactam in an ST11-KL64 Hypervirulent Klebsiella pneumoniae.

With the widespread clinical use of ceftazidime-avibactam (CZA), reports of resistance have increased continuously, posing immense threats to public health worldwide. In this study, we explored the underlying mechanisms leading to the development of CZA resistance in an ST11-KL64 hypervirulent Klebsiella pneumoniae CRE146 that harbored the blaKPC-228 gene. Twelve carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were isolated from the same patient, including K. pneumoniae CRE146. Whole genome sequencing (WGS), phylogenetic analysis, blaKPC gene cloning and pACYC-KPC construction assays were conducted to further explore the molecular mechanisms of CZA resistance. Quantitative siderophore production assay, string test, capsule quantification and Galleria mellonella in vivo infection model were applied to verify the level of pathogenicity of K. pneumoniae CRE146. This strain carried key virulence factors, iutA-iucABCD operon and rmpA gene. Compared to the wild-type KPC-2 carbapenemase, the novel KPC-228 enzyme exhibited a deletion of four amino acids in the Ω-loop (del_167-170_ELNS). In addition, the emergence of CZA resistance appeared to be associated with drug exposure, and we observed the in vivo evolution of wild-type KPC-2 to KPC-228 and then the reversion to its original wild-type KPC-2. The blaKPC-228 gene was located within the double IS26 flanking the ISKpn6-blaKPC-228-ISKpn27 core structure and carried on an IncFII/IncR-type plasmid. Notably, CRE146 exhibited high-level resistance to CZA (64/4 mg/L) but increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L) respectively. PACYC-KPC plasmids were constructed and expressed in K. pneumoniae ATCC13883. Compared to K. pneumoniae ATCC13883 harboring blaKPC-2, K. pneumoniae ATCC13883 harboring blaKPC-228 exhibited a high-level resistance to CZA (32/4 mg/L) and increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L). Interestingly, K. pneumoniae ATCC13883 harboring blaKPC-228 showed a significant decrease in their resistance to all β-lactamases tested except CZA and ceftazidime. In conclusion, we reported a novel KPC variant, KPC-228, in a clinical ST11-KL64 hypervirulent K. pneumoniae strain, which conferred CZA resistance, possibly through enhancing ceftazidime affinity and reducing avibactam binding. The blaKPC-228 can mutate back to blaKPC-2 under carbapenem pressure, which was very detrimental to clinical treatment. This strain carried both resistance and virulence genes, posing a major challenge in clinical management.

Antibacterial, antibiofilm, and gene expression assessment of ajwain (Trachyspermum ammi) essential oil on drug-resistant gastrointestinal pathogens and its combination effect with ampicillin.

Essential oils are natural substances used as therapeutic agents and food preservatives to inhibit harmful microorganisms. This study aimed to assess the synergistic effect of Trachyspermum ammi essential oil and ampicillin on antibiotic-resistant gastrointestinal pathogens, including Escherichia coli, Enterococcus faecalis, Shigella flexneri, and Salmonella serotype Typhimurium. Using gas chromatography-mass spectrometry (GC-MS), the main components of T. ammi essential oil were identified as thymol, gamma terpenes, and cymene. The antibacterial and antibiofilm properties were evaluated by minimum inhibitory concentration (MIC), disk diffusion, and microtiter plate methods, revealing MIC values of 2, 1, 4, and 4mg ml-1 for E. coli, E. faecalis, S. flexneri, and S. Typhimurium, respectively, and inhibition zones between 10 and 14mm. Pathogens were examined for their biofilm-related virulence genes, including aggR, esp, icsA, and fliC, using real-time polymerase chain reaction (RT-PCR) in E. coli, E. faecalis, S. flexneri, and S. Typhimurium, respectively. The methyl thiazole tetrazolium (MTT) assay was used to evaluate the essential oil's effect on the viability of human embryonic kidney 293 (HEK293) cells, which showed cell viability of over 80%. The combination of T. ammi oil and ampicillin demonstrated a synergistic effect, and biofilm formation was inhibited. E. faecalis exhibited the greatest sensitivity, while S. flexneri exhibited the lowest sensitivity.

Molecular investigation of an epidemic dissemination of vancomycin-resistant Enterococcus faecium sequence type 80 in Guangdong Province, China.

The detection rate of vancomycin-resistant Enterococcus faecium (VREfm) displayed a dramatically increase in Guangdong, China from 2021 to 2023, for which the molecular epidemiology and genomic characteristics remain largely unexplored. In this study, we investigated the genetic features and epidemiology of VREfm isolates in Guangdong. A total of 54 Guangdong VREfm isolates were collected from three tertiary hospitals in Guangdong. We preformed antimicrobial susceptibility test, whole genome sequencing, risk factor analysis, and bioinformatics analysis to conduct this research. Our investigation indicated that VREfm isolates were highly clonal and multidrug-resistant ST80 Enterococcus faecium harboring vanA-positive plasmid. The phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) demonstrated that VREfm isolates exhibited minimal genetic similarity to previously reported E. faecium in China, whereas exhibited high genomically similar to an India strain A10290 isolated in 2019 and Hiroshima isolates detected in 2020, indicative of a possible exogeneous import. The genetic environment of cps region showed a novel type of wzy gene cluster involved in capsule polysaccharide (CPS) biosynthesis flanked by ISEf1 and IS16 was identified in VREfm isolates, which displayed a remarkably divergence from the downstream of putative cpsABCD region in other sequence types (STs) E. faecium. Heat map of plasmid-mediated virulence factors suggested several predicted proteins including Cag pathogenetic island proteins existed in VREfm isolates at a high frequency. This study highlighted the importance of ongoing surveillance to track the dynamic dissemination of multidrug-resistant ST80 VREfm isolates harboring multiple virulence genes in Guangdong province, China.

Antimicrobial resistance and virulence factors analysis of a multidrug-resistant Acinetobacter baumannii isolated from chickens using whole-genome sequencing

Multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) is currently recognized not only as a significant nosocomial pathogen but also is an emerging bacterial infection in food-producing animals, posing a critical threat to global health. However, this is a hindrance to detailed bioinformatic studies of MDR A. baumannii of chicken origin due to the lack of its complete genome sequence. Here, we report whole-genome sequencing analysis of MDR A. baumannii Y03 isolated from chickens. The Y03 genome consists of 1 circular chromosome and 4 circular plasmids, The Y03 chromosome harbors 41 antimicrobial resistance genes conferring resistance to major classes of antibiotics, including β-lactams, phenicols, macrolides, lincosamides, aminoglycosides, and nitrofurans, as well as 135 virulence factors involved in effector delivery system, immune modulation, adherence, stress survival, biofilm, exotoxin, and nutritional/metabolic factor. The in vivo infection experiments certificated that Y03 was virulent to chickens. Meanwhile, we used PCR amplification method to detect 10 antimicrobial resistance genes including abeM, adeB, adeH, adeK, blaapmC, blaOXA−90, catB9, macB, folP, and parE, as well as 14 virulence genes including lpxC, pilO, fimT, ompA, basA, bauA, gspL, csu, pgaC, plc2, tssA, tviB, bap, and vgrG. Whole-genome sequencing analysis revealed that Y03 contained 46 horizontal gene transfer elements, including 11 genomic islands, 30 transposons, and 5 prophages, as well as 518 mutations associated with reduced virulence and 44 mutations resulting in loss of pathogenicity. Furthermore, there were 22 antibiotic targets and 28 lethal mutations on the Y03 chromosome that could be used as potential targets to prevent, control, and treat infections caused by MDR A. baumannii Y03. Therefore, this study contributes to the development of strategies for the prevention, control, and treatment of A. baumannii infections and their spread in chickens.

Molecular Characterization of Streptococcus spp. Isolated from Milk, Feces and Farm Environment of Mastitic Dairy Cows

Streptococci are the primary cause of mastitis, significantly impacting the dairy industry economically. This study aimed to explore the molecular epidemiology, antimicrobial resistance, and virulence characteristics of Streptococcus spp. isolated from the milk, feces, and environment of dairy cows with mastitis. Sixty (60) samples (milk = 20, feces = 20 and soil = 20) from cows with clinical mastitis (CM) were purposively collected and examined. Samples were enriched in Luria Bertani broth (LB) and Streptococcus spp. was isolated on Modified Edwards Medium and confirmed by ribosomal (16S rRNA) gene sequencing. Twenty-two (36.67%) of the samples were positive for Streptococcus spp. (milk = 40.90%, feces = 31.82% and soil = 27.28%) by cultural and molecular examination. Phylogenetic analysis revealed 59.5, 30.5, 6.0% and 4.0 % of the Streptococcus isolates as S. uberis, S. agalactiae, S. hyovaginalis and S. urinalis, respectively. The milk samples had higher prevalence (42.75%) of S. uberis mastitis than feces (36.80%) and soil (20.45%) samples. Likewise, S. agalactiae was prevalent in 51.5%, 37.60% and 10.90% milk, feces and soil samples, respectively. The draft assembly sizes of G2M6 and G6M1 were 1,960,858 and 2,303,841 base pair (bp), respectively. The strains were typed as S. uberis sequence type 155 (ST155) and S. agalactiae sequence type 58 (ST58). The G2M6 genome contained 367 SEED subsystem features, 45 antimicrobial resistance genes (ARGs), and 160 virulence and virulence-related genes, whereas the G6M1 genome possessed 378 SEED subsystem features, 41 ARGs and 178 virulence genes. One plasmid replicon such as IncY of 4,012 bp, was identified in the G6M1 genome (with 95% identity and 60% coverage) while G2M6 genome harbored none. Genome completeness analysis using BUSCO revealed the presence of 100% complete BUSCOs in the hybrid assembly of both genomes. Streptococcus spp. associated with bovine mastitis exhibit a variety of genomic traits and harbor an array of ARGs and virulence genes. Further investigation is needed to identify specific traits that govern virulence fitness in the pathophysiology of mastitis. J. of Sci. and Tech. Res. 6(1): 01-12, 2024

Whole-Genome Sequencing and Genome Annotation of Pathogenic Elsinoë batatas Causing Stem and Foliage Scab Disease in Sweet Potato.

A pathogen strain responsible for sweet potato stem and foliage scab disease was isolated from sweet potato stems. Through a phylogenetic analysis based on the rDNA internal transcribed spacer (ITS) region, combined with morphological methods, the isolated strain was identified as Elsinoë batatas. To comprehensively analyze the pathogenicity of the isolated strain from a genetic perspective, the whole-genome sequencing of E. batatas HD-1 was performed using both the PacBio and Illumina platforms. The genome of E. batatas HD-1 is about 26.31 Mb long in 167 scaffolds, with a GC content of 50.81%, and 7898 protein-coding genes, 131 non-coding RNAs, and 1954 interspersed repetitive sequences were predicted. Functional annotation revealed that 408 genes encode virulence factors involved in plant disease (DFVF-Plant). Notably, twenty-eight of these virulence genes encode secretory carbohydrate-active enzymes (CAZymes), including two endo-1,4-β-xylanase genes and seven cutinase genes, which suggested that endo-1,4-β-xylanase and cutinase play a vital role in the pathogenicity of E. batatas HD-1 within sweet potato. In total, twelve effectors were identified, including five LysM effectors and two CDIP effectors, suggesting that LysM and CDIP effectors play significant roles in the interaction between E. batatas HD-1 and sweet potato. Additionally, our analysis of biosynthetic gene clusters (BGCs) showed that two gene clusters are involved in melanin and choline metabolism. This study enriches the genomic resources of E. batatas and provides a theoretical foundation for future investigations into the pathogenic mechanisms of its infection in sweet potatoes, as well as potential targets for disease control.

Characterization of a novel covS SNP identified in Australian group A Streptococcus isolates derived from the M1UK lineage.

Group A Streptococcus (GAS) is a human-adapted pathogen responsible for a variety of diseases. The GAS M1UK lineage has contributed significantly to the recently reported increases in scarlet fever and invasive infections. However, the basis for its evolutionary success is not yet fully understood. During the transition to systemic disease, the M1 serotype is known to give rise to spontaneous mutations in the control of virulence two-component regulatory system (CovRS) that confer a fitness advantage during invasive infections. Mutations that inactivate CovS function result in the de-repression of key GAS virulence factors such as streptolysin O (SLO), a pore-forming toxin and major trigger of inflammasome/interleukin-1β-dependent inflammation. Conversely, expression of the streptococcal cysteine protease SpeB, which is required during initial stages of colonization and onset of invasive disease, is typically lost in such mutants. In this study, we identified and characterized a novel covS single nucleotide polymorphism detected in three separate invasive M1UK isolates. The resulting CovSAla318Val mutation caused a significant upregulation of SLO resulting in increased inflammasome activation in human THP-1 macrophages, indicating an enhanced inflammatory potential. Surprisingly, SpeB production was unaffected. Site-directed mutagenesis was performed to assess the impact of this mutation on virulence and global gene expression. We found that the CovSAla318Val mutation led to subtle, virulence-specific changes of the CovRS regulon compared to previously characterized covS mutations, highlighting an unappreciated level of complexity in CovRS-dependent gene regulation. Continued longitudinal surveillance is warranted to determine whether this novel covS mutation will expand in the M1UK lineage.IMPORTANCEThe M1UK lineage of GAS has contributed to a recent global upsurge in scarlet fever and invasive infections. Understanding how GAS can become more virulent is critical for infection control and identifying new treatment approaches. The two-component CovRS system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence genes. In the M1 serotype, covRS mutations are associated with an invasive phenotype. Such mutations have not been fully characterized in the M1UK lineage. This study identified a novel covS mutation in invasive Australian M1UK isolates that resulted in a more nuanced virulence gene regulation compared to previously characterized covS mutations. A representative isolate displayed upregulated SLO production and triggered amplified interleukin-1β secretion in infected human macrophages, indicating an enhanced inflammatory potential. These findings underscore the need for comprehensive analyses of covRS mutants to fully elucidate their contribution to M1UK virulence and persistence.

Characteristics of phage-plasmids and their impact on microbial communities.

Bacteria host various foreign genetic elements, most notably plasmids and bacteriophages (or phages). Historically, these two classes were seen as separate, but recent research has shown considerable interplay between them. Phage-plasmids (P-Ps) exhibit characteristics of both phages and plasmids, allowing them to exist extrachromosomally within bacterial hosts as plasmids, but also to infect and lyse bacteria as phages. This dual functionality enables P-Ps to utilize the modes of transmission of both phage and plasmids, facilitating the rapid dissemination of genetic material, including antibiotic resistance and virulence genes, throughout bacterial populations. Additionally, P-Ps have been found to encode toxin-antitoxin and CRISPR-Cas adaptive immune systems, which enhance bacterial survival under stress and provide immunity against other foreign genetic elements. Despite a growing body of literature on P-Ps, large gaps remain in our understanding of their ecological roles and environmental prevalence. This review aims to synthesise existing knowledge and identify research gaps on the impacts of P-Ps on microbial communities.

High-Risk Lineages of Hybrid Plasmids Carrying Virulence and Carbapenemase Genes

Background/Objectives: Carbapenem-resistant Enterobacterales (CRE) are a global health threat due to their high morbidity and mortality rates and limited treatment options. This study examines the plasmid-mediated transmission of virulence and antibiotic resistance determinants in carbapenem-resistant Klebsiella pneumoniae (Kpn) and Escherichia coli (E. coli) isolated from Russian hospitals. Methods: We performed short- and long-read whole-genome sequencing of 53 clinical isolates (48 Kpn and 5 E. coli) attributed to 15 genetic lineages and collected from 21 hospitals across nine Russian cities between 2016 and 2022. Results: The plasmid analysis identified 18 clusters that showed high concordance with replicon typing, with all clusters having a major replicon type. The majority of plasmids in the IncHI1B(pNDM-MAR)/IncFIB(pNDM-Mar)-like cluster (79.16%) carried both antibiotic resistance genes (e.g., blaNDM-1 and blaOXA-48) and virulence factors (VFs) such as siderophore genes. We hypothesized that hybrid plasmids could play a critical role in the dissemination of antibiotic resistance genes and VFs. Comparative analyses with global plasmid databases revealed high-risk lineages of hybrid plasmids that are predominantly spread throughout Russia at present. Conclusions: Our findings underscore the importance of monitoring plasmid backbones for clinical management, surveillance, and infection control activities.