Effect of mucoid property on antibiotic susceptibility ofPseudomonas aeruginosa

Clinical isolates ofPseudomonas aeruginosa from patients with cystic fibrosis (CF) were examined for susceptibility to the antibiotics carbenicillin, ticarcillin, tobramycin, gentamicin, and tetracycline. Minimal inhibitory concentrations of the antibiotics were determined for mucoid and nonmucoid isolates from the same patient by a single-colony replica plating method. This method allows the rapid determination of antibiotic susceptibility of a single cell’s progeny and the individual screening of each colony against all antibiotics. Twenty of 34 (58%) cystic fibrosis patients had a mucoid isolate which was more susceptible to antibiotics than their nonmucoid isolate of the same serotype. Nonmucoid revertant segregants of mucoid strains isolated from 50% of the patients demonstrated greater resistance to at least one antibiotic than the original mucoid strain. Multiple isolates from 25 patients were serotyped by Difco (Liu) or Homma antiserum; only 2 patients harbored multiple strains with no common serotyping antigens. Serotypes of the nonmucoid revertants were the same as the original mucoid isolate even if the susceptibilities of the two strains were not similar.     

The Role of Fluoroquinolones in the Promotion of Alginate Synthesis and Antibiotic Resistance in Pseudomonas aeruginosa

Treatment of nonmucoid Pseudomonas aeruginosa with gyrase inhibitors such as ciprofloxacin, norfloxacin, and ofloxacin, which target the A subunit of topoisomerase II, resulted in 100% conversion to the mucoid phenotype. However, antibiotics that partially inhibited growth and macromolecular synthesis (DNA, RNA, protein, or peptidoglycan) of nonmucoid isolates in a gluconate-limited chemostat culture system did not promote conversion to mucoid subpopulations. An increase in resistance was observed in populations that expressed the mucoid phenotype. Both mucoid conversion and antibiotic resistance were completely reversible when ciprofloxacin pressure was withdrawn, but only partially reversible by the removal of norfloxacin and ofloxacin. Thus, these experiments indicate that in the presence of some fluoroquinolones, a conditional response resulting in mucoid conversion and antibiotic resistance may occur. Received: 28 January 1997 / Accepted: 12 February 1997     

Enzyme activities and antibiotic susceptibility of colonial variants of Bacillus subtilis and Bacillus licheniformis.

A nonmucoid colonial variant of a mucoid Bacillus subtilis strain produced less amylase activity and a transparent colonial variant of a B. licheniformis strain produced less protease activity compared with their parents. Antibiotic susceptibility patterns of the colonial variants differed, and increased resistance to beta-lactam antibiotics was correlated with increased production of extracellular beta-lactamase.     

Enzyme activities and antibiotic susceptibility of colonial variants of Bacillus subtilis and Bacillus licheniformis

A nonmucoid colonial variant of a mucoid Bacillus subtilis strain produced less amylase activity and a transparent colonial variant of a B. licheniformis strain produced less protease activity compared with their parents. Antibiotic susceptibility patterns of the colonial variants differed, and increased resistance to beta-lactam antibiotics was correlated with increased production of extracellular beta-lactamase.     

In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.     

Swimming Motility in a Longitudinal Collection of Clinical Isolates of Burkholderia cepacia Complex Bacteria from People with Cystic Fibrosis

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.     

Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.     

Adherence of clinical isolates ofPseudomonas aeruginosa to hamster trachael epithelium in vitro

The adherence to hamster tracheal epithelium, of mucoid and nonmucoid clinical isolates ofPseudomonas aeruginosa from cystic fibrosis patients, was studied using tracheal organ cultures. Tracheal cultures were infected with 10⁷ colony-forming units per ml of either mucoid or nonmucoid clinical isolates ofP aeruginosa. The tracheal explants were rinsed at various time intervals to remove nonadherent bacteria, fixed, and prepared for transmission-and scanning-electron microscopy. Mucoid isolates were seen adhering to the ciliated epithelium as early as 4 h after initiation of infection, whereas nonmucoid isolates were only observed adhering at 6 to 8 h after infection. Mucoid organisms were found as clusters of bacteria embedded in an extensive extracellular matrix. The nonmucoid isolates were generally found as single organisms with no evidence of an extracellular matrix. These results suggest that the prevalence of mucoid isolates ofP. aeruginosa in cystic fibrosis may be due to adherent properties of the mucoid organism.     

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild‐type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild‐types attained similar population densities. Given the importance of the methyl‐directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild‐type phenotype.     

Morphotypic Conversion in Listeria monocytogenes Biofilm Formation: Biological Significance of Rough Colony Isolates

Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.     

Identification of a slime exopolysaccharide depolymerase in mucoid strains ofPseudomonas aeruginosa

Strains ofPseudomonas aeruginosa recovered from pulmonary infections in cystic fibrosis (CF) patients are often mucoid in appearance owing to the secretion of a viscous slime exopolysaccharide (EPS). Unlike most mucoid isolates, strains WcM#2, P10, and P11 produce mucoid colonies after 24 h of incubation at 37°C, which become nonmucoid upon further incubation; this suggests the presence of a slime-degrading enzyme or depolymerase. Using both qualitative and quantitative assays, the presence of a slime EPS depolymerase was confirmed in each of these three strains as well as in four of four additional mucoid strains. Depolymerase activity was lower but still detectable in four of four nonmucoid strains. Enzyme preparations from strains WcM#2, P10, and P11 were active on most, but not all, slime EPS preparations fromP. aeruginosa strains, as well as sodium alginate; greater activity was observed on substrates after deacetylation. Comparisons are made between the enzyme described in this study and previous reports of slime EPS depolymerase in mucoid strains ofP. aeruginosa.     

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ²²). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.     

A genetic analysis system of Burkholderia cepacia: construction of mobilizable transposons and a cloning vector

A genetic analysis system of Burkholderia cepacia (Bc) was developed which included transposon mutagenesis and complementation of mutation with the cloned genes of interest. To deliver the transposon in this multidrug-resistant microorganism, two plasmids, pKN30 and pKN31, were constructed which contained Tn5 derivatives, Tn5-30Tp and Tn5-31Tp, respectively, carrying Km^R and Tp^R genes. The plasmids have the origin of ColE1 replication and the mobilization gene of RP4. Tn5-31Tp was mobilized to Bc KF1, a strain isolated from a pneumonia patient, by the transfer system of RP4 integrated in the chromosome of Escherichia coli (Ec). Selection with trimethoprim resulted in generation of a number of transposants of Bc KF1. Fourteen protease-deficient mutants were isolated, all of which contained a single transposon marker in the chromosome. Thirteen protease-deficient mutants were also lipase deficient. An Ec-Bc shuttle plasmid, pTS1209, was constructed that consists of oriColE1, oripSa, Ap^R and Cm^R genes, and several unique restriction sites for cloning. Plasmid pTS1209 was successfully employed for cloning genes of Bc involved in protease production.     

Impact of Alginate Overproduction on Attachment and Biofilm Architecture of a Supermucoid Pseudomonas aeruginosa Strain

The supermucoid Pseudomonas aeruginosa strain PDO300Δalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.Alginate is an important virulence factor for Pseudomonas aeruginosa, and the conversion of nonmucoid strains to alginate-overproducing mucoid strains early after the infection of cystic fibrosis patients is associated with a decline of pulmonary function and survival rate (11, 13). Alginate functions as extracellular matrix material, enabling the formation of differentiated biofilms in which the diffusion of clinical antibiotics is decreased and the embedded cells are protected against human antibacterial defense mechanisms (9, 12). Although alginate is not required for P. aeruginosa biofilm formation (15), previous studies have provided evidence that it plays a role in the formation of thick and three-dimensional biofilms (5, 9). To further investigate the impact of alginate on attachment and biofilm architecture, we used a recently generated supermucoid strain, PDO300Δalg8(pBBR1MCS-5:alg8) (14). This strain showed about 15-fold alginate overproduction compared to alginate-producing mucoid P. aeruginosa. The gene alg8 encodes the proposed catalytic subunit of alginate polymerase and is essential for alginate biosynthesis (14).     

A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/U activity results in the loss of alginate production

Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg(+)) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg(+) due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.     

Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics

Bacterial opportunistic human pathogens frequently exhibit intrinsic antibiotic tolerance and resistance, resulting in infections that can be nearly impossible to eradicate. We asked whether this recalcitrance could be driven by these organisms’ evolutionary history as environmental microbes that engage in chemical warfare. Using Pseudomonas aeruginosa as a model, we demonstrate that the self-produced antibiotic pyocyanin (PYO) activates defenses that confer collateral tolerance specifically to structurally similar synthetic clinical antibiotics. Non-PYO-producing opportunistic pathogens, such as members of the Burkholderia cepacia complex, likewise display elevated antibiotic tolerance when cocultured with PYO-producing strains. Furthermore, by widening the population bottleneck that occurs during antibiotic selection and promoting the establishment of a more diverse range of mutant lineages, PYO increases apparent rates of mutation to antibiotic resistance to a degree that can rival clinically relevant hypermutator strains. Together, these results reveal an overlooked mechanism by which opportunistic pathogens that produce natural toxins can dramatically modulate the efficacy of clinical antibiotics and the evolution of antibiotic resistance, both for themselves and other members of clinically relevant polymicrobial communities.

This study shows that pyocyanin, a toxin secreted by the opportunistic pathogen Pseudomonas aeruginosa, induces defense responses that decrease the efficacy of structurally-similar clinical antibiotics and accelerate the evolution of antibiotic resistance, both in the producer and in other members of clinically-relevant polymicrobial communities.