Antibiotic Cycling Reverts Extensive Drug Resistance in Burkholderia multivorans.
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ABSTRACT:
SUBMITTER: Kavanaugh LG
PROVIDER: S-EPMC8284468 | biostudies-literature | 2021 Jul
REPOSITORIES: biostudies-literature
Publications
Antibiotic Cycling Reverts Extensive Drug Resistance in Burkholderia multivorans.
Kavanaugh L G LG Harrison S K SK Flanagan J N JN Steck T R TR
Antimicrobial agents and chemotherapy 20210716 8
Antibiotic collateral sensitivity, in which acquired resistance to one drug leads to decreased resistance to a different drug, occurs in Burkholderia multivorans. Here, we observed that treatment of extensively drug-resistant variants evolved from a cystic fibrosis (CF) sputum sample isolate with either meropenem or sulfamethoxazole-trimethoprim, depending on past resistance phenotypes, resulted in increased sensitivity to five different classes of antibiotics. We further identified mutations, i ...[more]
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Project description:Antibiotic collateral sensitivity (CS) occurs when a bacterium that acquires resistance to a treatment drug exhibits decreased resistance to a different drug. Here we identify reciprocal CS networks and candidate genes in Burkholderia multivorans. Burkholderia multivorans was evolved to become resistant to each of six antibiotics. The antibiogram of the evolved strain was compared with the immediate parental strain to determine CS and cross-resistance. The evolution process was continued for each resistant strain. CS interactions were observed in 170 of 279 evolved strains. CS patterns grouped into two clusters based on the treatment drug being a β-lactam antibiotic or not. Reciprocal pairs of CS antibiotics arose in ≥25% of all evolved strains. A total of 68 evolved strains were subjected to whole-genome sequencing and the resulting mutation patterns were correlated with antibiograms. Analysis revealed there was no single gene responsible for CS and that CS seen in B. multivorans is likely due to a combination of specific and non-specific mutations. The frequency of reciprocal CS, and the degree to which resistance changed, suggests a long-term treatment strategy; when resistance to one drug occurs, switch to use of the other member of the reciprocal pair. This switching could theoretically be continued indefinitely, allowing life-long treatment of chronic infections with just two antibiotics.
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Project description:Burkholderia multivorans is a member of the Burkholderia cepacia complex whose members are inherently resistant to many antibiotics and can cause chronic lung infections in patients with cystic fibrosis. A possible treatment for chronic infections arises from the existence of collateral sensitivity (CS)-acquired resistance to a treatment antibiotic results in a decreased resistance to a nontreatment antibiotic. Determining CS patterns for bacteria involved in chronic infections may lead to sustainable treatment regimens that reduce development of multidrug-resistant bacterial strains. CS has been found to occur in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Here, we report that B. multivorans exhibits antibiotic CS, as well as cross-resistance (CR), describe CS and CR networks for six antibiotics (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole), and identify candidate genes involved in CS. Characterization of CS and CR patterns allows antibiotics to be separated into two clusters based on the treatment drug to which the evolved strain developed primary resistance, suggesting an antibiotic therapy strategy of switching between members of these two clusters.
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Project description:Burkholderia multivorans is a member of the Burkholderia cepacia complex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. ?-Lactam antibiotics are recommended as therapy for infections due to Bmultivorans, which possesses two ?-lactamase genes, blapenA and blaAmpC PenA is a carbapenemase with a substrate profile similar to that of the Klebsiella pneumoniae carbapenemase (KPC); in addition, expression of PenA is inducible by ?-lactams in Bmultivorans Here, we characterize AmpC from Bmultivorans ATCC 17616. AmpC possesses only 38 to 46% protein identity with non-Burkholderia AmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates of Bmultivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (? loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward ?-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (Kiapp > 600 ?M), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible in Bmultivorans ATCC 17616 after growth in subinhibitory concentrations of imipenem (1 ?g/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role in Bmultivorans compared to PenA, which is the dominant ?-lactamase in Bmultivorans ATCC 17616.
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Project description:Can we exploit our burgeoning understanding of molecular evolution to slow the progress of drug resistance? One role of an infection clinician is exactly that: to foresee trajectories to resistance during antibiotic treatment and to hinder that evolutionary course. But can this be done at a hospital-wide scale? Clinicians and theoreticians tried to when they proposed two conflicting behavioral strategies that are expected to curb resistance evolution in the clinic, these are known as "antibiotic cycling" and "antibiotic mixing." However, the accumulated data from clinical trials, now approaching 4 million patient days of treatment, is too variable for cycling or mixing to be deemed successful. The former implements the restriction and prioritization of different antibiotics at different times in hospitals in a manner said to "cycle" between them. In antibiotic mixing, appropriate antibiotics are allocated to patients but randomly. Mixing results in no correlation, in time or across patients, in the drugs used for treatment which is why theorists saw this as an optimal behavioral strategy. So while cycling and mixing were proposed as ways of controlling evolution, we show there is good reason why clinical datasets cannot choose between them: by re-examining the theoretical literature we show prior support for the theoretical optimality of mixing was misplaced. Our analysis is consistent with a pattern emerging in data: neither cycling or mixing is a priori better than the other at mitigating selection for antibiotic resistance in the clinic.Key words: antibiotic cycling, antibiotic mixing, optimal control, stochastic models.
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Project description:BackgroundAlthough semi-selective growth media have been developed for the isolation of Burkholderia cepacia complex bacteria from the environment, thus far Burkholderia multivorans has rarely been isolated from such samples. Because environmental B. multivorans isolates mainly originate from water samples, we hypothesized that water rather than soil is its most likely environmental niche. The aim of the present study was to assess the occurrence of B. multivorans in water samples from Flanders (Belgium) using a fast, culture-independent PCR assay.ResultsA nested PCR approach was used to achieve high sensitivity, and specificity was confirmed by sequencing the resulting amplicons. B. multivorans was detected in 11 % of the water samples (n = 112) and 92 % of the soil samples (n = 25) tested. The percentage of false positives was higher for water samples compared to soil samples, showing that the presently available B. multivorans recA primers lack specificity when applied to the analysis of water samples.ConclusionsThe results of the present study demonstrate that B. multivorans DNA is commonly present in soil samples and to a lesser extent in water samples in Flanders (Belgium).
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Project description:Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.
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Project description:Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans and B. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of the B. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.
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Project description:Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.
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Project description:Antibiotic cycling has been proposed as a promising approach to slow down resistance evolution against currently employed antibiotics. It remains unclear, however, to which extent the decreased resistance evolution is the result of collateral sensitivity, an evolutionary trade-off where resistance to one antibiotic enhances the sensitivity to the second, or due to additional effects of the evolved genetic background, in which mutations accumulated during treatment with a first antibiotic alter the emergence and spread of resistance against a second antibiotic via other mechanisms. Also, the influence of antibiotic exposure patterns on the outcome of drug cycling is unknown. Here, we systematically assessed the effects of the evolved genetic background by focusing on the first switch between two antibiotics against Salmonella Typhimurium, with cefotaxime fixed as the first and a broad variety of other drugs as the second antibiotic. By normalizing the antibiotic concentrations to eliminate the effects of collateral sensitivity, we demonstrated a clear contribution of the evolved genetic background beyond collateral sensitivity, which either enhanced or reduced the adaptive potential depending on the specific drug combination. We further demonstrated that the gradient strength with which cefotaxime was applied affected both cefotaxime resistance evolution and adaptation to second antibiotics, an effect that was associated with higher levels of clonal interference and reduced cost of resistance in populations evolved under weaker cefotaxime gradients. Overall, our work highlights that drug cycling can affect resistance evolution independently of collateral sensitivity, in a manner that is contingent on the antibiotic exposure pattern.
| S-EPMC9778841 | biostudies-literature
Project description:Burkholderia multivorans comprises opportunistic bacteria infecting hosts such as cystic fibrosis (CF) patients. Bcc long-term infection of CF patient airways has been associated with emergence of phenotypic variation including the mucoid-to-nonmucoid colony morphotype. Here we studied three Burkholderia multivorans clonal isolates (mucoid D2095 (BM11L), and nonmucoid D2214G and D2214P) and a laboratory nonmucoid variant (BM11L-nmv1) obtained under prolonged stationary phase. The aim is to identify possible traits associated to the different morphotypes and find possible mechanisms for this morphotypic variation.
2018-06-22 | GSE111467 | GEO