RpoS Acts as a Global Repressor of Virulence Gene Expression in Escherichia coli O104:H4 and Enteroaggregative E coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E coli (EAEC)-specific virulence factors. Here, we identified an E coli O104:H4 isolate that carried a single-nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

Proteus mirabilis UreR coordinates cellular functions required for urease activity.

A hallmark of Proteus mirabilis infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of ions to form stones. Transcriptional regulator UreR activates expression of urease genes in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant. Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from one of two nickel transporters and facilitate incorporation of nickel to form mature urease. In this study, we performed a comprehensive RNA-seq to define the P. mirabilis urea-induced transcriptome as well as the UreR regulon. We identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for the direct regulation of the Ynt nickel transporter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Our study demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis. IMPORTANCE: Catheter-associated urinary tract infections (CAUTIs) account for over 40% of acute nosocomial infections in the USA and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. Transcriptional regulation of urease genes by UreR is well established; here, we expand the UreR regulon to include regulation of nickel import, a function required to produce mature urease. Furthermore, we reflect on the role of urea catalysis in P. mirabilis metabolism and provide evidence for its importance.

AefR, a TetR Family Transcriptional Repressor, Regulates Several Auxin Responses in Pseudomonas syringae Strain PtoDC3000.

The plant hormone indole-3-acetic acid (IAA), also known as auxin, plays important roles in plant growth and development, as well as in several plant-microbe interactions. IAA also acts as a microbial signal and in many bacteria regulates metabolism, stress responses, and virulence. In the bacterial plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000), exposure to IAA results in large-scale transcriptional reprogramming, including the differential expression of several known virulence genes. However, how PtoDC3000 senses and responds to IAA and what aspects of its biology are regulated by IAA is not understood. To investigate the mechanisms involved in perceiving and responding to IAA, we carried out a genetic screen for mutants with altered responses to IAA. One group of mutants of particular interest carried disruptions in the aefR gene encoding a TetR family transcriptional regulator. Gene expression analysis confirmed that the aefR mutants have altered responses to IAA. Thus, AefR is the first demonstrated auxin response regulator in PtoDC3000. We also investigated several aspects of PtoDC3000 biology that are regulated by both AefR and IAA, including antibiotic resistance, motility, and virulence. The observation that the aefR mutant has altered virulence on Arabidopsis, suggests that the sector of the IAA response regulated by aefR is important during pathogenesis. Our findings also provide evidence that AefR plays a role in coordinating changes in gene expression during the transition from early to late stages of infection. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genomic investigation of Salmonella enterica Serovar Welikade from a pediatric diarrhea case first time in Shanghai, China.

BACKGROUND: Salmonella, an important foodborne pathogen, was estimated to be responsible for 95.1 million cases and 50,771 deaths worldwide. Sixteen serovars were responsible for approximately 80% of Salmonella infections in humans in China, and infections caused by a few uncommon serovars have been reported in recent years, though not with S. Welikade. This study reports the first clinical case caused by S. Welikade in China and places Chinese S. Welikade isolates in the context of global isolates via genomic analysis. For comparison, S. Welikade isolates were also screened in the Chinese Local Surveillance System for Salmonella (CLSSS). The minimum inhibitory concentrations (MICs) of 28 antimicrobial agents were determined using the broth microdilution method. The isolates were sequenced on an Illumina platform to identify antimicrobial resistance genes, virulence genes, and phylogenetic relationships. RESULTS: The S. Welikade isolate (Sal097) was isolated from a two-year-old boy with acute gastroenteritis in 2021. Along with the other two isolates found in CLSSS, the three Chinese isolates were susceptible to all the examined antimicrobial agents, and their sequence types (STs) were ST5123 (n = 2) and ST3774 (n = 1). Single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed that global S. Welikade strains can be divided into four groups, and these three Chinese isolates were assigned to B (n = 2; Sal097 and XXB1016) and C (n = 1; XXB700). In Group B, the two Chinese ST5123 isolates were closely clustered with three UK ST5123 isolates. In Group C, the Chinese isolate was closely related to the other 12 ST3774 isolates. The number of virulence genes in the S. Welikade isolates ranged from 59 to 152. The galF gene was only present in Group A, the pipB2 gene was only absent from Group A, the avrA gene was only absent from Group B, and the allB, sseK1, sspH2, STM0287, and tlde1 were found only within Group C and D isolates. There were 15 loci unique to the Sal097 isolate. CONCLUSION: This study is the first to characterize and investigate clinical S. Welikade isolates in China. Responsible for a pediatric case of gastroenteritis in 2021, the clinical isolate harbored no antimicrobial resistance and belonged to phylogenetic Group B of global S. Welikade genomes.

Exploring Nocardia's ecological spectrum and novel therapeutic frontiers through whole-genome sequencing: unraveling drug resistance and virulence factors.

Nocardia farcinica is the leading pathogen responsible for nocardiosis, a life-threatening infection primarily affecting immunocompromised patients. In this study, the genomic sequence of a clinically isolated N. farcinica sample was sequenced. Subsequently, the assembled genome was annotated to identify antimicrobial resistance and virulence genes, as well as plasmid and prophages. The analysis of the entire genome size was 6,021,225 bp, with a GC content of 70.78% and consists of 103 contigs and N50 values of 292,531 bp. The genome analysis revealed the presence of several antimicrobial resistance genes, including RbpA, mtrA, FAR-1, blaFAR-1, blaFAR-1_1, and rox. In addition, virulence genes such as relA, icl, and mbtH were also detected. The present study signifies that N. farcinica genome is pivotal for the understanding of antimicrobial resistance and virulence genes is crucial for comprehending resistance mechanism, and developing effective strategies to combat bacterial infections effectively, especially adhesins and toxins. This study aids in identifying crucial drug targets for combating multidrug-resistant N. farcinica in the future.

Investigation and characterization of Aliarcobacter spp. isolated from cattle slaughterhouse in Türkiye.

Aliarcobacter spp. have been isolated from numerous food products at retail and from animal carcasses and feces at slaughter. The objectives of this study were as follows: (i) to isolate Aliarcobacter species from different slaughterhouses' samples and (ii) to detect genetic diversity, antibiotic resistance, biofilm ability, and putative virulence gene profiles of the isolates. A molecular investigation of antibiotic resistance and virulence factors was also conducted using polymerase chain reaction (PCR). Among 150 samples, a total of 22 (14.6%) Aliarcobacter spp. isolates were obtained, with varying levels of antibiotic resistance observed. The genes tetO, tetW, and gyrA were detected in 0%, 31.8%, and 27.2% of the isolates, respectively. All isolates were resistant to ampicillin, rifampin, and erythromycin, while tetracycline was found to be the most effective antibiotic, with 81.8% of the isolates showing susceptibility to it. All isolates (100%) harbored more than one of the nine putative virulence genes tested, with 18.1% of isolates carrying more than three. Regarding biofilm formation, 7 (31.8%) and 4 (18.1%) isolates were found to form strong and moderate biofilms, respectively, while one (4.5%) isolate was classified as a weak biofilm producer. ERIC-PCR band patterns suggested that the isolated Aliarcobacter spp. from slaughterhouses had different sources of contamination. These findings highlight the potential risk posed by pathogenic and multidrug-resistant Aliarcobacter spp. in food and the need for control measures throughout the food chain to prevent the spread of these strains. The results indicate that foods of animal origin and cattle slaughterhouses are significant sources of antimicrobial resistant Aliarcobacter.

N-glycosylation of the envelope glycoprotein I is essential for the proliferation and virulence of the duck plague virus.

Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N69, N78, and N265) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease.

Some virulence genes are associated with antibiotic susceptibility in Enterobacter cloacae complex.

BACKGROUND: Enterobacter cloacae complex (ECC) including different species are isolated from different human clinical samples. ECC is armed by many different virulence genes (VGs) and they were also classified among ESKAPE group by WHO recently. The present study was designed to find probable association between VGs and antibiotic susceptibility in different ECC species. METHODS: Forty-five Enterobacter isolates that were harvested from different clinical samples were classified in four different species. Seven VGs were screened by PCR technique and antibiotic susceptibility assessment was performed by disk-diffusion assay. RESULT: Four Enterobacter species; Enterobacter cloacae (33.3%), Enterobacter hormaechei (55.6%), Enterobacter kobei (6.7%) and Enterobacter roggenkampii (4.4%) were detected. Minimum antibiotic resistance was against carbapenem agents and amikacin even in MDR isolates. 33.3% and 13.3% of isolates were MDR and XDR respectively. The rpoS (97.8%) and csgD (11.1%) showed maximum and minimum frequency respectively. Blood sample isolated were highly virulent but less resistant in comparison to the other sample isolates. The csgA, csgD and iutA genes were associated with cefepime sensitivity. CONCLUSION: The fepA showed a predictory role for differentiating of E. hormaechei from other species. More evolved iron acquisition system in E. hormaechei was hypothesized. The fepA gene introduced as a suitable target for designing novel anti-virulence/antibiotic agents against E. hormaechei. Complementary studies on other VGs and ARGs and with bigger study population is recommended.

Tuber pathogenicity of Macrophomina phaseolina strain 3 a isolated from rotten cassava tuber from farm lands in Osogbo, Osun State, Nigeria, its virulence genes and ADMET properties.

BACKGROUND: Macrophomina phaseolina is a pathogen that causes an opportunistic disease that spreads by soil and seeds and affects more than 500 different plant species, like fruits, trees, and row crops. Mycotoxins, such as phaseolinic acid, and phaseolinone, are produced by M. phaseolina isolates in previous investigations; however, the production of these mycotoxins seems to vary depending on the host and the region. METHODS AND RESULTS: In this study, Macrophomina phaseolina strain 3 A was isolated from rotten cassava tuber and identified using the analysis of the sequences of the internal transcribed spacer region. The isolate was inoculated on a fresh healthy cassava tuber at 25 °C and tuber-rotting potential was monitored for 4 weeks. Virulence genes MPH_06603, MPH_06955, and MPH_01521 were determined with designed primers, and secondary metabolites were characterized by FTIR and GCMS. The rotten tuber effect was observed from the 2nd week of the experiment with severe tuber rot and weight reduction. The PCR showed the presence of MPH_06603 virulence gene. The GCMS showed N-Methylpivalamide (115.0 m/z), Butane, 1,4-dimethoxy- (119.0 m/z), and 5-Hydroxymethylfurfural (126.0 m/z) were the predominant metabolites produced by the pathogen. The compounds in the metabolites inhibit CYP3A4 enzymes, cause eye irritation, and Human Ether-a-go-go-related gene inhibition. CONCLUSION: This study revealed that M. phaseolina was responsible for the cassava tuber rot which leads to a lower yield of farm produce. The metabolites produced are toxic and unsafe for human consumption. It is suggested that farmers should destroy any cassava affected by this pathogen to prevent its toxic effects on humans and animals.

Phylogenetic analysis, biofilm formation, antimicrobial resistance and relationship between these characteristics in Uropathogenic Escherichia coli.

BACKGROUND: In the present study, we examine the prevalence of phylogenetic groups, O-serogroups, adhesin genes, antimicrobial resistance, the level of gene expression associated with biofilm formation, and the presence of extended-spectrum beta-lactamase (ESBL) in UPEC strains isolated from both pediatric and adult patients. METHODS: In this cross-sectional study, 156 UPEC isolates were collected from UTI patients. ESBL-producing isolates were detected using the double-disc synergy (DDS) method, and biofilm formation was assessed through a microplate assay. The presence of O-serogroups, adhesion factors and resistance genes, including ESBLs and PMQR genes, was detected by PCR, and isolates were categorized into phylogenetic groups using multiplex PCR. Additionally, the quantitative real-time PCR method was also used to determine the expression level of genes related to biofilm. RESULTS: During the study period, 50.6% (79/156) of the samples were obtained from children, and 49.4% (77/156) were from adults. The highest rate of resistance was to NA (91.7%), while FM (10.9%) had the lowest rate of antibiotic resistance. In addition, 67.9% (106/156) of UPEC isolates were ESBL producers. Most of UPEC isolates belonged to phylogenetic group B2 (37.1%). This study revealed that blaCTX-M and qnrS are widely distributed among UPEC isolates. The mean expression levels of fimA genes were significantly higher in non-biofilm producers than in biofilm producers (p < 0.01). CONCLUSIONS: The high antibiotic resistance rates in this study highlight the significance of local resistance monitoring and investigating underlying mechanisms. Our findings indicate the dominance of phylogroup B2 and group D as the prevailing phylogenetic groups. Consequently, it is imperative to investigate the epidemiological aspects and characterize UPEC isolates across diverse regions and time frames.

Preliminary comparative genomics analysis among Corynebacterium kroppenstedtii complex necessitates a reassessment of precise species associated with mastitis.

AIMS: This study aimed to characterize the first complete genome of Corynebacterium parakroppenstedtii and clarify the evolutionary relationship in the Corynebacterium kroppenstedtii complex (CKC) by using comparative genomics analysis. METHODS AND RESULTS: The genome of isolate yu01 from a breast specimen was sequenced, and 35 CKC genomes were collected. Analysis of 16S rRNA, rpoB, and fusA suggested ambiguous identification, whereas ANI analysis assigned isolate yu01 as Coryne. parakroppenstedtii. The fourth genospecies "Corynebacterium aliikroppenstedtii" was identified in CKC. Comparative genomics analysis suggested that the genomic arrangement in CKC was highly conserved. A total of 43 potential virulence genes and 79 species-specific genes were detected. Most genome-based phylogenetic analysis were incapable of resolving the interspecific evolutionary relationships among CKCs. A total of 20 core genes were found to be distinguishable in CKC. CONCLUSIONS: This study suggested the limited divergence and unavailability of normal single gene-based identification in CKC and questioned the precise species of strains associated with mastitis, identified as Coryne. kroppenstedtii in previous studies. The 20 genes showed potential to enhance the methods for the identification and epidemiological investigation of CKC.

Pathomolecular epidemiology, antimicrobial resistance, and virulence genes of Streptococcus dysgalactiae subsp. equisimilis isolates from slaughtered pigs in India.

AIMS: We aimed to investigate the prevalence, pathology, and characterization of Streptococcus dysgalactiae subsp. equisimilis (SDSE) in slaughtered pigs of India. METHODS AND RESULTS: We collected 1254 morbid tissues (lungs-627 and spleen-627) and 627 heart-blood from 627 slaughtered pigs. The bacterial isolation, antibiogram, virulence gene profiling, and mouse pathogenicity testing were performed for the detection and characterization of SDSE. A total of 177 isolates (heart-blood-160 and tissues-17) were recovered from 627 slaughtered pigs with higher isolation rate in heart-blood (25.51%). The prevalence of SDSE was 11% in morbid tissues by polymerase chain reaction. Majority of isolates showed higher detection of streptolysin O, followed by streptokinase and extracellular phospholipase A virulence genes with higher degree of resistance to azithromycin, clindamycin, erythromycin, and penicillin antibiotics. Mouse pathogenicity testing confirmed virulence based on histopathological lesions and re-isolation of SDSE. CONCLUSIONS: Our findings highlight the high prevalence of SDSE in slaughtered pigs. The presence of virulence genes and mouse pathogenicity testing confirm their pathogenic potential.

Microbial organic fertilizer prepared by co-composting of Trichoderma dregs mitigates dissemination of resistance, virulence genes, and bacterial pathogens in soil and rhizosphere.

The use of manure, mycelium dregs and other waste as organic fertilizer is the main source of antibiotic resistance genes (ARGs) and pathogens in farmland. Composting of waste may effectively remove ARGs and pathogens. However, the profiles and drivers of changes in metal resistance genes (MRGs), biocide resistance genes (BRGs), and virulence genes (VGs) in soil-crop rhizosphere systems after compost application remain largely unknown. Here, we prepared two kinds of microbial organic fertilizers (MOF) by using Trichoderma dregs (TDs) and organic fertilizer mixing method (MOF1) and TDs co-composting method (MOF2). The effects of different types and doses of MOF on resistance genes, VGs and pathogens in soil-rhizosphere system and their potential mechanisms were studied. The results showed that co-composting of TDs promoted the decomposition of organic carbon and decreased the absolute abundance of ARGs and mobile genetic elements (MGEs) by 53.4-65.0%. MOF1 application significantly increased the abundance and diversity of soil ARGs, BRGs, and VGs, while low and medium doses of MOF2 significantly decreased their abundance and diversity in soil and rhizosphere. Patterns of positive co-occurrence between MGEs and VGs/MRGs/BRGs/ARGs were observed through statistical analysis and gene arrangements. ARGs/MRGs reductions in MOF2 soil were directly driven by weakened horizontal gene transfer triggered by MGEs. Furthermore, MOF2 reduced soil BRGs/VGs levels by shifting bacterial communities (e.g., reduced bacterial host) or improving soil property. Our study provided new insights into the rational use of waste to minimize the spread of resistomes and VGs in soil.

Phenotypic Characteristics, Antimicrobial Susceptibility and Virulence Genotype Features of Trueperella pyogenes Associated with Endometritis of Dairy Cows.

Trueperella pyogenes can cause various infections in the organs and tissues of different livestock (including pigs, cows, goats, and sheep), including mastitis, endometritis, pneumonia, or abscesses. Moreover, diseases induced by T. pyogenes cause significant economic losses in animal husbandry. In recent large-scale investigations, T. pyogenes has been identified as one of the main pathogens causing endometritis in lactating cows. However, the main treatment for the above-mentioned diseases is still currently antibiotic therapy. Understanding the impact of endometritis associated with T. pyogenes on the fertility of cows can help optimize antibiotic treatment for uterine diseases, thereby strategically concentrating the use of antimicrobials on the most severe cases. Therefore, it is particularly important to continuously monitor the prevalence of T. pyogenes and test its drug resistance. This study compared the uterine microbiota of healthy cows and endometritis cows in different cattle farms, investigated the prevalence of T. pyogenes, evaluated the genetic characteristics and population structure of isolated strains, and determined the virulence genes and drug resistance characteristics of T. pyogenes. An amount of 186 dairy cows were involved in this study and 23 T. pyogenes strains were isolated and identified from the uterine lavage fluid of dairy cows with or without endometritis.

Oxidative Stress-Mediated Repression of Virulence Gene Transcription and Biofilm Formation as Antibacterial Action of Cinnamomum burmannii Essential Oil on Staphylococcus aureus.

This work aimed to identify the chemical compounds of Cinnamomum burmannii leaf essential oil (CBLEO) and to unravel the antibacterial mechanism of CBLEO at the molecular level for developing antimicrobials. CBLEO had 37 volatile compounds with abundant borneol (28.40%) and showed good potential to control foodborne pathogens, of which Staphylococcus aureus had the greatest inhibition zone diameter (28.72 mm) with the lowest values of minimum inhibitory concentration (1.0 µg/mL) and bactericidal concentration (2.0 µg/mL). To unravel the antibacterial action of CBLEO on S. aureus, a dynamic exploration of antibacterial growth, material leakage, ROS formation, protein oxidation, cell morphology, and interaction with genome DNA was conducted on S. aureus exposed to CBLEO at different doses (1/2-2×MIC) and times (0-24 h), indicating that CBLEO acts as an inducer for ROS production and the oxidative stress of S. aureus. To highlight the antibacterial action of CBLEO on S. aureus at the molecular level, we performed a comparative association of ROS accumulation with some key virulence-related gene (sigB/agrA/sarA/icaA/cidA/rsbU) transcription, protease production, and biofilm formation in S. aureus subjected to CBLEO at different levels and times, revealing that CBLEO-induced oxidative stress caused transcript suppression of virulence regulators (RsbU and SigB) and its targeted genes, causing a protease level increase destined for the biofilm formation and growth inhibition of S. aureus, which may be a key bactericidal action. Our findings provide valuable information for studying the antibacterial mechanism of essential oil against pathogens.

[Virulence genes and antimicrobial resistance of Bacillus cereus in prepackaged pastries sold in Taizhou from 2020 to 2022].

OBJECTIVE: To investigate the virulence genes and antimicrobial resistance of Bacillus cereus from the pre-packaged pastries in Taizhou city. METHODS: 500 pre-packaged patries were collected in taizhou city market. 97 Bacillus cereus strains were detected from them by GB 4789.14-2014 method and identified with 4 houseking genes, then 13 virulence genes were detected by polymerase chain reaction(PCR)method and the antimicrobial resistance of Bacillus cereus to 19 antibiotics was detected by paper diffusion method. RESULTS: The result showed that the contamination rate of Bacillus cereus was 19.4% in 500 pre-packaged pastries. The detection rate of four housekeeping genes groEL, gyr B, rpoB and Vrr were 100%, 94.8%, 97.9% and 96.9%, respectively, and 89.7% at the same time. The virulence gene test result showed that the detection rate of nheABC, entFM, bceT, cytK and hblABCD were 91.8%, 88.7%, 61.9%, 51.6% and 25.8%, emetic virulence genes had the lowest detection rate, ces and EMl were 4.1%, cer was 5.2%. 97 Bacillus cereus strains show different degrees of drug resistance to 14 antimicrobials, the resistance rates to penicillin, ampicillin, cefotaxime and cotrimoxazole were higher than 95%, but they were completely sensitive to streptomycin, vancomycin and chloramphenicol. CONCLUSION: There is a risk of contamination by diarrhea-type Bacillus cereus and vomiting-type Bacillus cereus in prepackaged pastries in Taizhou. The isolated and identified Bacillus cereus has multiple-drug resistance.

Relationship between virulence and carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection: identification of a carbapenem-resistant and hypervirulent strain. / è¡€æ¶²æ„ŸæŸ“æ¥æºè‚ºç‚Žå ‹é›·ä¼¯èŒæ¯’åŠ›ä¸Žç¢³é’éœ‰çƒ¯ç±»è€è¯è¡¨åž‹çš„å ³ç³»åŠç¢³é’éœ‰çƒ¯ç±»è€è¯é«˜æ¯’åŠ›èŒæ ªçš„é‰´å®š.

OBJECTIVES: To investigate the relationship between the virulence and the carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection, and to identify carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HVKP)strains. METHODS: A total of 192 Klebsiella pneumoniae strains were isolated from blood culture of patients with bloodstream infections from 2016 to 2019, of which 96 isolates were carbapenem-resistant Klebsiella pneumoniae (CRKP) and 96 were carbapenem-sensitive Klebsiella pneumoniae (CSKP). The drug susceptibility was detected by VITEK-2 automatic microbial analyzer; carbapenemase genes, virulence genes and capsule typing were detected by polymerase chain reaction; the high viscosity phenotype of strains was detected by string test, and the genome characteristics of CR-HVKP were detected by whole genome sequencing. Serum killing and biofilm formation test were used to further verify the virulence of CR-HVKP. RESULTS: There were significant differences in drug resistance to common antibiotics, except for minocycline between CSKP and CRKP isolates (all P<0.05). 92 out of 96 CRKP isolates carried carbapenemase genes, mainly blaKPC-2. The string tests were positive in 4 isolates of CRKP and 36 isolates of CSKP (P<0.05). The detection rates of virulence genes Kfu, aerobictin, iutA, ybtS, rmpA, magA, allS, and capsule antigen K1 and K2 in CSKP group were significantly higher than those in CRKP group (all P<0.05). One HVKP strain was detected in the CRKP group (CR-HVKP) and 36 HVKP was detected in the CSKP group (P<0.05). The CR-HVKP strain belonged to the MLST412, serotype K57, expressed iutA, entB, mrkD, fimH, and rmpA virulence genes, and showed strong biofilm formation and significantly increased serum resistance. Whole genome sequencing results showed that this CR-HVKP isolate carried blaSHV-145, blaTEM-1, blaCTX-M-3, fosA6, oqxA5, oqxB26, and aac(3)-IId resistance genes, accompanied by abnormalities in outer membrane protein K (OmpK) 35 and OmpK36. CONCLUSIONS: The drug resistance of CRKP is significantly higher than that of CSKP, while CRKP carrying fewer virulence genes in both number and types compared to CSKP. A new MLST type of carbapenem-resistant and hypervirulent Klebsiella pneumoniae strain has been detected, which requires clinical awareness and epidemiological monitoring.

Transcription Factor PecS Mediates Agrobacterium fabrum Fitness and Survival.

The transcriptional regulator PecS is encoded by select bacterial pathogens. For instance, in the plant pathogen Dickeya dadantii, PecS controls a range of virulence genes, including pectinase genes and the divergently oriented gene pecM, which encodes an efflux pump through which the antioxidant indigoidine is exported. In the plant pathogen Agrobacterium fabrum (formerly named Agrobacterium tumefaciens), the pecS-pecM locus is conserved. Using a strain of A. fabrum in which pecS has been disrupted, we show here that PecS controls a range of phenotypes that are associated with bacterial fitness. PecS represses flagellar motility and chemotaxis, which are processes that are important for A. fabrum to reach plant wound sites. Biofilm formation and microaerobic survival are reduced in the pecS disruption strain, whereas the production of acyl homoserine lactone (AHL) and resistance to reactive oxygen species (ROS) are increased when pecS is disrupted. AHL production and resistance to ROS are expected to be particularly relevant in the host environment. We also show that PecS does not participate in the induction of vir genes. The inducing ligands for PecS, urate, and xanthine, may be found in the rhizosphere, and they accumulate within the plant host upon infection. Therefore, our data suggest that PecS mediates A. fabrum fitness during its transition from the rhizosphere to the host plant. IMPORTANCE PecS is a transcription factor that is conserved in several pathogenic bacteria, where it regulates virulence genes. The plant pathogen Agrobacterium fabrum is important not only for its induction of crown galls in susceptible plants but also for its role as a tool in the genetic manipulation of host plants. We show here that A. fabrum PecS controls a range of phenotypes, which would confer the bacteria an advantage while transitioning from the rhizosphere to the host plant. This includes the production of signaling molecules, which are critical for the propagation of the tumor-inducing plasmid. A more complete understanding of the infection process may inform approaches by which to treat infections as well as to facilitate the transformation of recalcitrant plant species.

Genomic characterization and comparative genomic analysis of HS-associated Pasteurella multocida serotype B:2 strains from Pakistan.

BACKGROUND: Haemorrhagic septicaemia (HS) is a highly fatal and predominant disease in livestock, particularly cattle and buffalo in the tropical regions of the world. Pasteurella multocida (P. multocida), serotypes B:2 and E:2, are reported to be the main causes of HS wherein serotype B:2 is more common in Asian countries including Pakistan and costs heavy financial losses every year. As yet, very little molecular and genomic information related to the HS-associated serotypes of P. multocida isolated from Pakistan is available. Therefore, this study aimed to explore the characteristics of novel bovine isolates of P. multocida serotype B:2 at the genomic level and perform comparative genomic analysis of various P. multocida strains from Pakistan to better understand the genetic basis of pathogenesis and virulence. RESULTS: To understand the genomic variability and pathogenomics, we characterized three HS-associated P. multocida serotype B:2 strains isolated from the Faisalabad (PM1), Peshawar (PM2) and Okara (PM3) districts of Punjab, Pakistan. Together with the other nine publicly available Pakistani-origin P. multocida strains and a reference strain Pm70, a comparative genomic analysis was performed. The sequenced strains were characterized as serotype B and belong to ST-122. The strains contain no plasmids; however, each strain contains at least two complete prophages. The pan-genome analysis revealed a higher number of core genes indicating a close resemblance to the studied genomes and very few genes (1%) of the core genome serve as a part of virulence, disease, and defense mechanisms. We further identified that studied P. multocida B:2 strains harbor common antibiotic resistance genes, specifically PBP3 and EF-Tu. Remarkably, the distribution of virulence factors revealed that OmpH and plpE were not present in any P. multocida B:2 strains while the presence of these antigens was reported uniformly in all serotypes of P. multocida. CONCLUSION: This study's findings indicate the absence of OmpH and PlpE in the analyzed P. multocida B:2 strains, which are known surface antigens and provide protective immunity against P. multocida infection. The availability of additional genomic data on P. multocida B:2 strains from Pakistan will facilitate the development of localized therapeutic agents and rapid diagnostic tools specifically targeting HS-associated P. multocida B:2 strains.

Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq.

Klebsiella pneumoniae is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent K. pneumoniae. The discovery and study of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern due to their restriction in the development of novel antibiotics. However, the lack of essential functional genomic data has hampered the study of the mechanisms of essential genes related to antimicrobial susceptibility. In this study, we developed a pooled CE genes mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference screening method (Mobile-CRISPRi-seq) for K. pneumoniae to identify genes that play critical roles in antimicrobial fitness in vitro and host immunity in vivo. Targeting 870 predicted CE genes in K. pneumoniae, Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes folB and folP under trimethoprim pressure. Our screening also identified genes waaE and fldA related to polymyxin and ß-lactam susceptibility by applying a screening strategy based on Mobile-CRISPRi-seq and comparative genomics. Furthermore, using a mouse infection model and Mobile-CRISPRi-seq, multiple virulence genes were identified, and among these genes, pal, yciS, and ribB were demonstrated to contribute to the pathogenesis of K. pneumoniae. This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in K. pneumoniae, and this broadly applicable system can be expanded for high-throughput functional gene study in multiple pathogenic bacteria, especially in gram-negative bacteria. IMPORTANCE The discovery and investigation of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern because of the restriction of antimicrobial targets in the development of novel antibiotics. In this study, we developed a pooled CE gene-wide mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference sequencing (Mobile-CRISPRi-seq) strategy in Klebsiella pneumoniae to identify genes that play critical roles in the fitness of antimicrobials in vitro and host immunity in vivo. The data suggest a robust tool to screen for loss-of-function phenotypes in a pooled gene knockdown library in K. pneumoniae, and Mobile-CRISPRi-seq may be expanded to multiple bacteria for screening and identification of genes with crucial roles in the fitness of antimicrobials and hosts.