Sub-inhibitory concentrations of vancomycin prevent quinolone-resistance in a penicillin-resistant isolate of Streptococcus pneumoniae

BACKGROUND: The continuous spread of penicillin-resistant pneumococci represents a permanent threat in the treatment of pneumococcal infections, especially when strains show additional resistance to quinolones. The main objective of this study was to determine a treatment modality impeding the emergence of quinolone resistance. RESULTS: Exposure of a penicillin-resistant pneumococcus to increasing concentrations of trovafloxacin or ciprofloxacin selected for mutants resistant to these drugs. In the presence of sub-inhibitory concentrations of vancomycin, development of trovafloxacin-resistance and high-level ciprofloxacin-resistance were prevented. CONCLUSIONS: Considering the risk of quinolone-resistance in pneumococci, the observation might be of clinical importance.

Efficacy of gatifloxacin alone and in combination with cefepime against penicillin-resistant Streptococcus pneumoniae in a rabbit meningitis model and in vitro

Gatifloxacin penetrated well into cerebrospinal fluid (CSF) (49 +/- 11%), measured by comparison of AUC(CSF)/AUC(serum), and showed good bactericidal activity (leading to a decrease of 0.75 +/- 0.17 log10 cfu/mL/h) in the treatment of experimental meningitis in rabbits caused by a penicillin-resistant pneumococcal strain (MIC 4 mg/L). It was significantly more effective than the standard regimen, ceftriaxone with vancomycin, which led to a decrease of 0.53 +/- 0.17 log10 cfu/mL/h. The addition of cefepime to gatifloxacin slightly improved the killing rates (giving a decrease of 0.84 +/- 0.14 log10 cfu/mL/h). In vitro, synergy was demonstrated between cefepime and gatifloxacin by the chequerboard method (fractional inhibitory concentration index = 0.5) and by viable counts over 8 h.

Pneumonia due to resistant Streptococcus pneumoniae

In the past 10 to 20 years the pneumococcus, the most common pathogen of community-acquired pneumonia, has developed resistance to most antibiotics used for its treatment. Classes with important resistance problems include the beta-lactams, the macrolides and lincosamides, trimethoprim-sulfamethoxazole, and the tetracyclines. Unfortunately, resistance to more than one class of antibiotics is common in pneumococci, and their treatment is thus becoming more difficult. Patients likely to harbour resistant organisms include young children, particularly those attending day care, older patients, and subjects who have received recent antibiotic therapy, suffer from underlying diseases including HIV, or have nosocomial or polymicrobial pneumonia. The consequences of resistance development are different for different classes of antibiotics. With beta-lactams, the increase in minimal inhibitory concentrations is usually moderate in resistant strains, and because of the high concentrations that can be achieved with this class of drugs resistance does not usually lead to treatment failure. Thus, beta-lactams continue to be important drugs for the treatment of pneumococcal pneumonia, even if the organism is resistant. In contrast, resistance to other classes of antibiotics must be assumed to render these drugs ineffective. Newer quinolones represent valuable alternatives for the treatment of pneumococcal pneumonia, since their efficacy is not affected by resistance to other classes of antibiotics and they cover almost all pathogens of community-acquired pneumonia, including the atypical pathogens. However, they should be used with restraint in order to preserve this valuable class of drugs.

Trovafloxacin in treatment of rabbits with experimental meningitis caused by high-level penicillin-resistant Streptococcus pneumoniae

The fluoroquinolone trovafloxacin was bactericidal (0.47 +/- 0.23 delta log10 CFU/ml x h after 10 mg/kg of body weight and 0.78 +/- 0.15 delta log10 CFU/ml x h after 30 mg/kg) in the treatment of experimental meningitis caused by a highly penicillin-resistant (MIC and minimum bactericidal concentration = 4 and 4 microg/ml) strain of Streptococcus pneumoniae. Combinations with ampicillin and rifampin were indifferent compared to single drugs.

Toxicity of Streptococcus pneumoniae in neurons, astrocytes, and microglia in vitro

The toxicity of pneumococci and endotoxin in primary cultures of rat neurons, astrocytes, and microglia and in a human astrocyte and two human glial cell lines was determined. Heat-inactivated, rough pneumococci (up to 10(8) cfu/mL) or their cell wall (up to 50 micrograms/mL) produced dose-dependent toxicity after 48 h in microglial cells and to a lesser extent in astrocytes but not in neurons. Toxicity was similar for equivalent doses of heat-inactivated organisms and pneumococcal cell wall, but time-course experiments showed significant differences between the two stimuli. Endotoxin at concentrations of up to 5 micrograms/mL did not induce significant toxicity in any of the cells. Thus, pneumococci can induce toxicity in two brain cell types, microglia and astrocytes, and the pneumococcal cell wall appears to mediate toxicity. Direct toxic effects of bacteria on brain cells may in part be responsible for brain injury during meningitis.

Differences of pathophysiology in experimental meningitis caused by three strains of Streptococcus pneumoniae

Differences in cytochemical and pathophysiologic abnormalities in experimental meningitis caused by pneumococcal strains A, B, and C were determined. Strain C produced the most severe abnormalities of cerebrospinal fluid (CSF) concentrations of lactate (P less than .01), protein (P less than .02), and glucose (P less than .01), CSF white blood cell count (P less than .04), cerebral blood flow (P less than .02), and clinical signs (P less than .05). Brain edema occurred only with strains A anc C, with no association with disease severity; intracranial hypertension was also independent of disease severity. Strain B, not C, achieved the highest bacterial titers in the CSF (P less than .005). The widely different abilities of strains of Streptococcus pneumoniae to induce intracranial abnormalities suggest that virulence determinants affect not only evasion of defense during colonization and invasion, as shown in other models, but also determine the course of disease once infection has been established. Differences of cell-wall metabolism among pneumococcal strains may play a role in this latter phase of the development of meningitis.

The postantibiotic effect in the treatment of experimental meningitis caused by Streptococcus pneumoniae in rabbits

The relevance of a postantibiotic effect in the treatment of pneumococcal meningitis was evaluated in a rabbit model. After administration of a single intravenous bolus of ampicillin at various dosages, such an effect was observed in all animals. The duration of this effect in vivo (2.5-18 hr) was consistently longer than that in vitro (1-4.3 hr); however, in rabbits the postantibiotic effect was eliminated by the administration of intravenous plus intracisternal beta-lactamase. In an assessment of the potential therapeutic benefit of the postantibiotic effect, the efficacy to two regimens of treatment with different intervals between doses was compared. One group of animals received ampicillin every 4 hr and another every 12 hr. With sufficiently high doses, drug concentrations in cerebrospinal fluid exceeded the minimal bactericidal concentration for most of the 4-hr interval but for only about one-third of the 12-hr interval. The rate of cure was similar for the two regimens and approximated 100% when peak drug concentrations in cerebrospinal fluid exceeded the minimal bactericidal concentration by at least 10-fold.

Antibacterial activity of beta-lactam antibiotics in experimental meningitis due to Streptococcus pneumoniae

In order to define the characteristics of the antibacterial activity of beta-lactam antibiotics in the treatment of bacterial meningitis, the relationship between cerebrospinal fluid (CSF) drug concentrations and the rate of bacterial killing was investigated for penicillin G and four new cephalosporins in an animal model of meningitis due to Streptococcus pneumoniae. All five drugs showed a significant correlation between increasing drug concentrations in CSF and increasing bactericidal rates. Minimal activity was observed in CSF at drug concentrations of approximately the broth minimal bactericidal concentration (MBC). Maximal activity occurred with CSF concentrations 10-30 times higher. In vitro tests did not reproduce the unique correlation of increasing drug concentrations and killing activity found in vivo. When evaluating new beta-lactam antibiotics for the treatment of bacterial meningitis, it is reasonable to establish a minimum standard of CSF drug concentrations of greater than or equal to 30 times the MBC against the infecting organism.

Detection of Streptococcus pneumoniae strain cocolonization in the nasopharynx

Colonization with more than one distinct strain of the same species, also termed cocolonization, is a prerequisite for horizontal gene transfer between pneumococcal strains that may lead to change of the capsular serotype. Capsule switch has become an important issue since the introduction of conjugated pneumococcal polysaccharide vaccines. There is, however, a lack of techniques to detect multiple colonization by S. pneumoniae strains directly in nasopharyngeal samples. Two hundred eighty-seven nasopharyngeal swabs collected during the prevaccine era within a nationwide surveillance program were analyzed by a novel technique for the detection of cocolonization, based on PCR amplification of a noncoding region adjacent to the pneumolysin gene (plyNCR) and restriction fragment length polymorphism (RFLP) analysis. The numbers of strains and their relative abundance in cocolonized samples were determined by terminal RFLP. The pneumococcal carriage rate found by PCR was 51.6%, compared to 40.0% found by culture. Cocolonization was present in 9.5% (10/105) of samples, most (9/10) of which contained two strains in a ratio of between 1:1 and 17:1. Five of the 10 cocolonized samples showed combinations of vaccine types only (n = 2) or combinations of nonvaccine types only (n = 3). Carriers of multiple pneumococcal strains had received recent antibiotic treatment more often than those colonized with a single strain (33% versus 9%, P = 0.025). This new technique allows for the rapid and economical study of pneumococcal cocolonization in nasopharyngeal swabs. It will be valuable for the surveillance of S. pneumoniae epidemiology under vaccine selection pressure.

Streptococcus pneumoniae detects and responds to foreign bacterial peptide fragments in its environment

Streptococcus pneumoniae is an important cause of bacterial meningitis and pneumonia but usually colonizes the human nasopharynx harmlessly. As this niche is simultaneously populated by other bacterial species, we looked for a role and pathway of communication between pneumococci and other species. This paper shows that two proteins of non-encapsulated S. pneumoniae, AliB-like ORF 1 and ORF 2, bind specifically to peptides matching other species resulting in changes in the pneumococci. AliB-like ORF 1 binds specifically peptide SETTFGRDFN, matching 50S ribosomal subunit protein L4 of Enterobacteriaceae, and facilitates upregulation of competence for genetic transformation. AliB-like ORF 2 binds specifically peptides containing sequence FPPQS, matching proteins of Prevotella species common in healthy human nasopharyngeal microbiota. We found that AliB-like ORF 2 mediates the early phase of nasopharyngeal colonization in vivo. The ability of S. pneumoniae to bind and respond to peptides of other bacterial species occupying the same host niche may play a key role in adaptation to its environment and in interspecies communication. These findings reveal a completely new concept of pneumococcal interspecies communication which may have implications for communication between other bacterial species and for future interventional therapeutics.

A low affinity penicillin-binding protein 2x is required for heteroresistance in Streptococcus pneumoniae

Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the minimal inhibitory concentration (MIC). This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin binding proteins PBP2b and PBP2x and secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from heteroresistant strain Spain(23F)2349 in non-heteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression is not observed and therefore expression does not correlate with an increased proportion of resistant subpopulations. Additional ciaR disruption mutants which have been described to mediate PBP-independent β-lactam resistance revealed no heteroresistant phenotype by PAP.We also showed, that the highly resistant subpopulations (HOM*) of transformants containing low affinity pbp2x undergo an increase in resistance upon selection on penicillin plates which partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins pstS, phoU, pstB and pstC in these highly resistant subpopulations.In conclusion, the presence of low affinity pbp2x enables certain pneumococcal colonies to survive in the presence of beta lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaption to antibiotic stress.

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence

BackgroundThe polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.ResultsHere, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.ConclusionsWe identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated Streptococcus pneumoniae (Non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if Non-Ec-Sp evolve sporadically, if they have high antibiotic non-susceptiblity rates and a unique, specific gene content. Here, whole genome sequencing of 131 Non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multi-locus sequences types ST344 (n=39) and ST448 (n=40). All ST344 and nine ST448 isolates had high non-susceptiblity rates to β-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic Non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic Non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic Non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P=0.005). In contrast, sporadic Non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, Non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of pneumococcal conjugate vaccines, Non-Ec-Sp may become more prevalent.

CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.