Interactions between the Prophage 919TP and Its Vibrio cholerae Host: Implications of gmd Mutation for Phage Resistance, Cell Auto-Aggregation, and Motility

Prophage 919TP is widely distributed among Vibrio cholera and is induced to produce free φ919TP phage particles. However, the interactions between prophage φ919TP, the induced phage particle, and its host remain unknown. In particular, phage resistance mechanisms and potential fitness trade-offs, resulting from phage resistance, are unresolved. In this study, we examined a prophage 919TP-deleted variant of V. cholerae and its interaction with a modified lytic variant of the induced prophage (φ919TP cI-). Specifically, the phage-resistant mutant was isolated by challenging a prophage-deleted variant with lytic phage φ919TP cI-. Further, the comparative genomic analysis of wild-type and φ919TP cI--resistant mutant predicted that phage φ919TP cI- selects for phage-resistant mutants harboring a mutation in key steps of lipopolysaccharide (LPS) O-antigen biosynthesis, causing a single-base-pair deletion in gene gmd. Our study showed that the gmd-mediated O-antigen defect can cause pleiotropic phenotypes, e.g., cell autoaggregation and reduced swarming motility, emphasizing the role of phage-driven diversification in V. cholerae. The developed approach assists in the identification of genetic determinants of host specificity and is used to explore the molecular mechanism underlying phage-host interactions. Our findings contribute to the understanding of prophage-facilitated horizontal gene transfer and emphasize the potential for developing new strategies to optimize the use of phages in bacterial pathogen control.

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Phage Resistance Is Associated with Decreased Virulence in KPC-Producing Klebsiella pneumoniae of the Clonal Group 258 Clade II Lineage

Microorganisms ◽

10.3390/microorganisms9040762 ◽

2021 ◽

Vol 9 (4) ◽

pp. 762

Author(s):

Lucia Henrici De Angelis ◽

Noemi Poerio ◽

Vincenzo Di Pilato ◽

Federica De Santis ◽

Alberto Antonelli ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Parental Strain ◽

Bacterial Infections ◽

Capsular Polysaccharide ◽

Galleria Mellonella ◽

Bacterial Pathogen ◽

Phage Resistance ◽

Infection Model ◽

Lytic Phage ◽

Susceptible Host

Phage therapy is now reconsidered with interest in the treatment of bacterial infections. A major piece of information for this application is the definition of the molecular targets exploited by phages to infect bacteria. Here, the genetic basis of resistance to the lytic phage φBO1E by its susceptible host Klebsiella pneumoniae KKBO-1 has been investigated. KKBO-1 phage-resistant mutants were obtained by infection at high multiplicity. One mutant, designated BO-FR-1, was selected for subsequent experiments, including virulence assessment in a Galleria mellonella infection model and characterization by whole-genome sequencing. Infection with BO-FR-1 was associated with a significantly lower mortality when compared to that of the parental strain. The BO-FR-1 genome differed from KKBO-1 by a single nonsense mutation into the wbaP gene, which encodes a glycosyltransferase involved in the first step of the biosynthesis of the capsular polysaccharide (CPS). Phage susceptibility was restored when BO-FR-1 was complemented with the constitutive wbaP gene. Our results demonstrated that φBO1E infects KKBO-1 targeting the bacterial CPS. Interestingly, BO-FR-1 was less virulent than the parental strain, suggesting that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host.

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Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa396 ◽

2020 ◽

Vol 76 (1) ◽

pp. 91-100

Author(s):

Jorge Arca-Suárez ◽

Cristina Lasarte-Monterrubio ◽

Bruno-Kotska Rodiño-Janeiro ◽

Gabriel Cabot ◽

Juan Carlos Vázquez-Ucha ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Mechanisms ◽

Genomic Analysis ◽

Resistance Mechanisms ◽

Comparative Genomic ◽

Development Of Resistance ◽

Structural Impact ◽

Genetic Context

Abstract Background The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. Objectives Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. Methods Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. Results The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. Conclusions Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.

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Pseudomonas donghuensis HYS gtrA/B/II Gene Cluster Contributes to Its Pathogenicity toward Caenorhabditis elegans

International Journal of Molecular Sciences ◽

10.3390/ijms221910741 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10741

Author(s):

Yaqian Xiao ◽

Panning Wang ◽

Xuesi Zhu ◽

Zhixiong Xie

Keyword(s):

Caenorhabditis Elegans ◽

Gene Cluster ◽

Mapk Pathway ◽

Genomic Analysis ◽

Virulence Gene ◽

Comparative Genomic ◽

Specific Gene ◽

C Elegans ◽

O Antigen ◽

Specific Virulence

Pseudomonas donghuensis HYS is more virulent than P. aeruginosa toward Caenorhabditis elegans but the mechanism underlying virulence is unclear. This study is the first to report that the specific gene cluster gtrA/B/II in P. donghuensis HYS is involved in the virulence of this strain toward C. elegans, and there are no reports of GtrA, GtrB and GtrII in any Pseudomonas species. The pathogenicity of P. donghuensis HYS was evaluated using C. elegans as a host. Based on the prediction of virulence factors and comparative genomic analysis of P. donghuensis HYS, we identified 42 specific virulence genes in P. donghuensis HYS. Slow-killing assays of these genes showed that the gtrAB mutation had the greatest effect on the virulence of P. donghuensis HYS, and GtrA, GtrB and GtrII all positively affected P. donghuensis HYS virulence. Two critical GtrII residues (Glu47 and Lys480) were identified in P. donghuensis HYS. Transmission electron microscopy (TEM) showed that GtrA, GtrB and GtrII were involved in the glucosylation of lipopolysaccharide (LPS) O-antigen in P. donghuensis HYS. Furthermore, colony-forming unit (CFU) assays showed that GtrA, GtrB and GtrII significantly enhanced P. donghuensis HYS colonization in the gut of C. elegans, and glucosylation of LPS O-antigen and colonization in the host intestine contributed to the pathogenicity of P. donghuensis HYS. In addition, experiments using the worm mutants ZD101, KU4 and KU25 revealed a correlation between P. donghuensis HYS virulence and the TIR-1/SEK-1/PMK-1 pathways of the innate immune p38 MAPK pathway in C. elegans. In conclusion, these results reveal that the specific virulence gene cluster gtrA/B/II contributes to the unique pathogenicity of HYS compared with other pathogenic Pseudomonas, and that this process also involves C. elegans innate immunity. These findings significantly increase the available information about GtrA/GtrB/GtrII-based virulence mechanisms in the genus Pseudomonas.

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Pleiotropy complicates a trade-off between phage resistance and antibiotic resistance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919888117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11207-11216 ◽

Cited By ~ 14

Author(s):

Alita R. Burmeister ◽

Abigail Fortier ◽

Carli Roush ◽

Adam J. Lessing ◽

Rose G. Bender ◽

...

Keyword(s):

Antibiotic Resistance ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Phage Resistance ◽

Resistance Mutations ◽

Trade Off ◽

Trade Offs ◽

Evolutionary Context ◽

Structural Barrier

Bacteria frequently encounter selection by both antibiotics and lytic bacteriophages. However, the evolutionary interactions between antibiotics and phages remain unclear, in particular, whether and when phages can drive evolutionary trade-offs with antibiotic resistance. Here, we describeEscherichia coliphage U136B, showing it relies on two host factors involved in different antibiotic resistance mechanisms: 1) the efflux pump protein TolC and 2) the structural barrier molecule lipopolysaccharide (LPS). Since TolC and LPS contribute to antibiotic resistance, phage U136B should select for their loss or modification, thereby driving a trade-off between phage resistance and either of the antibiotic resistance mechanisms. To test this hypothesis, we used fluctuation experiments and experimental evolution to obtain phage-resistant mutants. Using these mutants, we compared the accessibility of specific mutations (revealed in the fluctuation experiments) to their actual success during ecological competition and coevolution (revealed in the evolution experiments). BothtolCand LPS-related mutants arise readily during fluctuation assays, withtolCmutations becoming more common during the evolution experiments. In support of the trade-off hypothesis, phage resistance viatolCmutations occurs with a corresponding reduction in antibiotic resistance in many cases. However, contrary to the hypothesis, some phage resistance mutations pleiotropically confer increased antibiotic resistance. We discuss the molecular mechanisms underlying this surprising pleiotropic result, consideration for applied phage biology, and the importance of ecology in evolution of phage resistance. We envision that phages may be useful for the reversal of antibiotic resistance, but such applications will need to account for unexpected pleiotropy and evolutionary context.

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A Genomic Comparative Analysis of Bacillus Coagulans Unravels the Genetic Potential of MA-13 Strain for Biotechnological Applications

10.20944/preprints202112.0372.v1 ◽

2021 ◽

Author(s):

Martina Aulitto ◽

Laura Martinez-Alvarez ◽

Gabriella Fiorentino ◽

Danila Limauro ◽

Xu Peng ◽

...

Keyword(s):

Genomic Analysis ◽

Bacillus Coagulans ◽

Resistance Mechanisms ◽

Genomic Diversity ◽

Comparative Genomic ◽

Biotechnological Applications ◽

Secretion Systems ◽

Substrate Conversion ◽

Microbial Strains ◽

Environmental Robustness

The production of bio-chemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Bacillus coagulans spp. So far the genomes of about 50 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of CAZymes, secretion systems, and resistance mechanisms to environmental challenges. Moreover, B. coagulans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated Cas) genes, was also analysed. Overall, this study expands our understanding of the strains genomic diversity of B. coagulans to fully exploit its potential in biotechnological applications.

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Comparative Genomic Analysis of Fifty-Two Staphylococcus aureus Isolates Identified from Uncharacterized Staphylococcus Genomes in the NCBI database

10.1101/2020.06.18.159814 ◽

2020 ◽

Author(s):

Mohamed A. Abouelkhair

Keyword(s):

Staphylococcus Aureus ◽

Genome Sequence ◽

Food Poisoning ◽

Bacterial Pathogen ◽

Variant Calling ◽

Genomic Analysis ◽

Comparative Genomic ◽

Genome Sequences ◽

Spa Typing ◽

Genome Level

AbstractBackgroundStaphylococcus aureus is a major bacterial pathogen that causes a variety of diseases, ranging from wound infections to severe bacteremia or food poisoning. The course and severity of the disease are mainly dependent on the bacterium genotype as well as host factors. Whole-genome sequencing (WGS) is currently the most extensive genotyping method available, followed by bioinformatic sequence analysis.MethodsA total of 253 uncharacterized staphylococcus genome sequences were downloaded from the National Center for Biotechnology Information (NCBI) (August 2012 to March 2020) from different studies. Samples were clustered based on core and accessory pairwise distances between isolates and then analyzed by multilocus sequence typing tool (MLST). Staphylococcal Cassette Chromosome mec (SCCmec), spa typing, variant calling, core genome alignment, and recombination sites prediction were performed on detected S. aureus isolates. S. aureus isolates were also analyzed for the presence of genes coding for virulence factors and antibiotic resistance.Results and conclusionUncategorized genome sequences were clustered into 24 groups. About 182 uncharacterized Staphylococcus genomes were identified at the species level based on MLST, including 32 S. lugdunensis genome sequence, thus doubling the number of the publicly accessible S. lugdunensis genome sequence in Genbank. MLST identified another four species (S. epidermidis (33/253), S. lugdunensis (32/253), S. haemolyticus (41/253), S. hominis (24/253) and S. aureus (52/253)). Among the 52 S. aureus isolates, 21 (40.38%) isolates carried mecA gene, with 57.14% classified as SCCmec IV. The results of this study provide knowledge that facilitates evolutionary studies of staphylococcal species and other bacteria at the genome level.

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Genome-driven elucidation of phage-host interplay and impact of phage resistance evolution on bacterial fitness

The ISME Journal ◽

10.1038/s41396-021-01096-5 ◽

2021 ◽

Author(s):

Pawel Markwitz ◽

Cédric Lood ◽

Tomasz Olszak ◽

Vera van Noort ◽

Rob Lavigne ◽

...

Keyword(s):

Growth Rate ◽

Ecological Impact ◽

Resistance Mechanisms ◽

Large Deletion ◽

Carrier State ◽

Cross Resistance ◽

Phage Resistance ◽

Lytic Phage ◽

Bacterial Growth Rate ◽

Resistance Evolution

AbstractWhen considering the interactions between bacteriophages and their host, the issue of phage-resistance emergence is a key element in understanding the ecological impact of phages on the bacterial population. It is also an essential parameter for the implementation of phage therapy to combat antibiotic-resistant pathogens. This study investigates the phenotypic and genetic responses of five Pseudomonas aeruginosa strains (PAO1, A5803, AA43, CHA, and PAK) to the infection by seven phages with distinct evolutionary backgrounds and recognised receptors (LPS/T4P). Emerging phage-insensitivity was generally accompanied by self and cross-resistance mechanisms. Significant differences were observed between the reference PAO1 responses compared to other clinical representatives. LPS-dependent phage infections in clinical strains selected for mutations in the “global regulatory” and “other” genes, rather than in the LPS-synthesis clusters detected in PAO1 clones. Reduced fitness, as proxied by the growth rate, was correlated with large deletion (20–500 kbp) and phage carrier state. Multi-phage resistance was significantly correlated with a reduced growth rate but only in the PAO1 population. In addition, we observed that the presence of prophages decreased the lytic phage maintenance seemingly protecting the host against carrier state and occasional lytic phage propagation, thus preventing a significant reduction in bacterial growth rate.

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Comparative Genomic Analysis Provides Insights into the Phylogeny, Resistome, Virulome, and Host Adaptation in the Genus Ewingella

Pathogens ◽

10.3390/pathogens9050330 ◽

2020 ◽

Vol 9 (5) ◽

pp. 330 ◽

Cited By ~ 1

Author(s):

Zhenghui Liu ◽

Hongyan Sheng ◽

Benjamin Azu Okorley ◽

Yu Li ◽

Frederick Leo Sossah

Keyword(s):

Antibiotic Resistance ◽

Control Strategies ◽

Bacterial Pathogen ◽

Genomic Analysis ◽

Defense Responses ◽

Host Adaptation ◽

Comparative Genomic Analysis ◽

Effective Control ◽

Comparative Genomic ◽

Temperature Oxidation

Ewingella americana is a cosmopolitan bacterial pathogen that has been isolated from many hosts. Here, we sequenced a high-quality genome of E. americana B6-1 isolated from Flammulina filiformis, an important cultivated mushroom, performed a comparative genomic analysis with four other E. americana strains from various origins, and tested the susceptibility of B6-1 to antibiotics. The genome size, predicted genes, and GC (guanine-cytosine) content of B6-1 was 4.67 Mb, 4301, and 53.80%, respectively. The origin of the strains did not significantly affect the phylogeny, but mobile genetic elements shaped the evolution of the genus Ewingella. The strains encoded a set of common genes for type secretion, virulence effectors, CAZymes, and toxins required for pathogenicity in all hosts. They also had antibiotic resistance, pigments to suppress or evade host defense responses, as well as genes for adaptation to different environmental conditions, including temperature, oxidation, and nutrients. These findings provide a better understanding of the virulence, antibiotic resistance, and host adaptation strategies of Ewingella, and they also contribute to the development of effective control strategies.

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Comparative Genomic Analysis of the pPT23A Plasmid Family of Pseudomonas syringae

Journal of Bacteriology ◽

10.1128/jb.187.6.2113-2126.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 2113-2126 ◽

Cited By ~ 34

Author(s):

Youfu Zhao ◽

Zhonghua Ma ◽

George W. Sundin

Keyword(s):

Pseudomonas Syringae ◽

Bacterial Pathogen ◽

Genomic Analysis ◽

Complete Sequence ◽

Secretion System ◽

Comparative Genomic ◽

Type Iv ◽

Gene Acquisition ◽

Genes Encoding ◽

Type Ivb Secretion

ABSTRACT Members of the pPT23A plasmid family of Pseudomonas syringae play an important role in the interaction of this bacterial pathogen with host plants. Complete sequence analysis of several pPT23A family plasmids (PFPs) has provided a glimpse of the gene content and virulence function of these plasmids. We constructed a macroarray containing 161 genes to estimate and compare the gene contents of 23 newly analyzed and eight known PFPs from 12 pathovars of P. syringae, which belong to four genomospecies. Hybridization results revealed that PFPs could be distinguished by the type IV secretion system (T4SS) encoded and separated into four groups. Twelve PFPs along with pPSR1 from P. syringae pv. syringae, pPh1448B from P. syringae pv. phaseolicola, and pPMA4326A from P. syringae pv. maculicola encoded a type IVA T4SS (VirB-VirD4 conjugative system), whereas 10 PFPs along with pDC3000A and pDC3000B from P. syringae pv. tomato encoded a type IVB T4SS (tra system). Two plasmids encoded both T4SSs, whereas six other plasmids carried none or only a few genes of either the type IVA or type IVB secretion system. Most PFPs hybridized to more than one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors. The overall gene contents of individual PFPs were more similar among plasmids within each of the four groups based on T4SS genes; however, a number of genes, encoding plasmid-specific functions or hypothetical proteins, were shared among plasmids from different T4SS groups. The only gene shared by all PFPs in this study was the repA gene, which encoded sequences with 87 to 99% amino acid identityamong 25 sequences examined. We proposed a model to illustrate the evolution and gene acquisition of the pPT23A plasmid family. To our knowledge, this is the first such attempt to conduct a global genetic analysis of this important plasmid family.

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Two homologous Salmonella serogroup C1-specific genes are required for flagellar motility and cell invasion

BMC Genomics ◽

10.1186/s12864-021-07759-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Xiujuan Zhou ◽

Bin Liu ◽

Yanhong Liu ◽

Chunlei Shi ◽

Pina M. Fratamico ◽

...

Keyword(s):

Bacterial Pathogen ◽

Genomic Analysis ◽

Comparative Genomic ◽

Rna Seq ◽

Flagellar Motility ◽

Wild Type ◽

Bacterial Physiology ◽

Functional Relationships ◽

Double Deletion ◽

Bacterial Enumeration

Abstract Background Salmonella is a major bacterial pathogen associated with a large number of outbreaks of foodborne diseases. Many highly virulent serovars that cause human illness belong to Salmonella serogroup C1, and Salmonella ser. Choleraesuis is a prominent cause of invasive infections in Asia. Comparative genomic analysis in our previous study showed that two homologous genes, SC0368 and SC0595 in Salmonella ser. Choleraesuis were unique to serogroup C1. In this study, two single-deletion mutants (Δ0368 and Δ0595) and one double-deletion mutant (Δ0368Δ0595) were constructed based on the genome. All these mutants and the wild-type strain were subjected to RNA-Seq analysis to reveal functional relationships of the two serogroup C1-specific genes. Results Data from RNA-Seq indicated that deletion of SC0368 resulted in defects in motility through repression of σ28 in flagellar regulation Class 3. Consistent with RNA-Seq data, results from transmission electron microcopy (TEM) showed that flagella were not present in △0368 and △0368△0595 mutants resulting in both swimming and swarming defects. Interestingly, the growth rates of two non-motile mutants △0368 and △0368△0595 were significantly greater than the wild-type, which may be associated with up-regulation of genes encoding cytochromes, enhancing bacterial proliferation. Moreover, the △0595 mutant was significantly more invasive in Caco-2 cells as shown by bacterial enumeration assays, and the expression of lipopolysaccharide (LPS) core synthesis-related genes (rfaB, rfaI, rfaQ, rfaY, rfaK, rfaZ) was down-regulated only in the △0368△0595 mutant. In addition, this study also speculated that these two genes might be contributing to serotype conversion for Salmonella C1 serogroup based on their apparent roles in biosynthesis of LPS and the flagella. Conclusion A combination of biological and transcriptomic (RNA-Seq) analyses has shown that the SC0368 and SC0595 genes are involved in biosynthesis of flagella and complete LPS, as well as in bacterial growth and virulence. Such information will aid to revealing the role of these specific genes in bacterial physiology and evolution within the serogroup C1.

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