Bacteriostatic or bactericidal effect of linezolid against multiresistant Streptococcus pneumoniae

The nasopharynx plays a critical role as the reservoir of Streptococcus pneumoniae, including drug-resistant strains particularly in children attending day care centers. A total 58 nasopharyngeal, multiresistant isolates of S. pneumoniae collected from healthy pre-school children were susceptible to linezolid (MIC = 0.25-1 mg/l), irrespective of serotype and drug resistance pattern. The majority of them (about 94%) were sensitive to the bactericidal effect of linezolid with MBCs = 0.5-4 mg/l. One isolate was killed at 8 mg/l of linezolid, while two at higher concentrations of this antibiotic with MBCs 16 or 32 mg/l, suggesting tolerance of linezolid. BOX-PCR fingerprinting data imply that two linezolid-tolerant strains belonged to distinct clones. Linezolid tolerance was confirmed by monitoring the viability of these isolates during exposure to 4 or 20 mg/l of this antibiotic. The linezolid-tolerant strains were sensitive to the bactericidal effect of vancomycin. Linezolid tolerance in clinical isolates of pneumococci may represent a potential therapeutic risk, especially in infections in which bactericidal activity of drug is critical for eradication of bacteria.     

Variation in Streptococcus pneumoniae susceptibility to human antimicrobial peptides may mediate intraspecific competition

Streptococcus pneumoniae is a facultative pathogen inhabiting the nasopharynx of humans where it is exposed to a range of antimicrobial peptides (AMPs) of the innate immune response. It is possible therefore that the susceptibility of strains to AMPs plays a role in determining their ability to colonize, and furthermore, that AMPs could mediate competitive interactions between co-colonizing genotypes. However, little is known about patterns of natural variation in AMP susceptibility of S. pneumoniae, and it is unclear whether the susceptibilities of an isolate to multiple human AMPs are correlated. We tested this by characterizing the susceptibility of 31 S. pneumoniae natural isolates to human neutrophil peptide (HNP-1) (α-defensin) and LL-37 (cathelicidin). We observed significant variation in susceptibility between isolates to both AMPs, and in the majority of isolates, susceptibilities to HNP-1 and LL-37 were uncorrelated. Clinical isolates were more susceptible to AMPs than were carriage isolates. The polysaccharide capsule of S. pneumoniae is thought to protect cells against AMPs. However, serotype alone could not explain the observed variation in susceptibility suggesting that genetic background plays an equally important role. We tested directly whether AMPs could mediate competition between isolates using competition experiments in the presence and absence of AMPs. These experiments demonstrated that AMPs could indeed reverse the outcome of competition between selected isolates. AMP-mediated competition could therefore contribute to the maintenance of intraspecific genetic diversity in S. pneumoniae.     

Antibiotic resistance of Streptococcus pneumoniae isolated from patient with home care pneumonia

The monitoring of the resistance of Streptococcus pneumoniae, one of the most important causative agents of extrahospital pneumonia, has been carried out. The data on its sensitivity to antibacterial preparations, widely used in clinical practice, have been summarized.     

Antibiotic selection pressure and macrolide resistance in nasopharyngeal Streptococcus pneumoniae: a cluster-randomized clinical trial

Background

It is widely thought that widespread antibiotic use selects for community antibiotic resistance, though this has been difficult to prove in the setting of a community-randomized clinical trial. In this study, we used a randomized clinical trial design to assess whether macrolide resistance was higher in communities treated with mass azithromycin for trachoma, compared to untreated control communities.

Methods and Findings

In a cluster-randomized trial for trachoma control in Ethiopia, 12 communities were randomized to receive mass azithromycin treatment of children aged 1–10 years at months 0, 3, 6, and 9. Twelve control communities were randomized to receive no antibiotic treatments until the conclusion of the study. Nasopharyngeal swabs were collected from randomly selected children in the treated group at baseline and month 12, and in the control group at month 12. Antibiotic susceptibility testing was performed on Streptococcus pneumoniae isolated from the swabs using Etest strips. In the treated group, the mean prevalence of azithromycin resistance among all monitored children increased from 3.6% (95% confidence interval [CI] 0.8%–8.9%) at baseline, to 46.9% (37.5%–57.5%) at month 12 (p = 0.003). In control communities, azithromycin resistance was 9.2% (95% CI 6.7%–13.3%) at month 12, significantly lower than the treated group (p<0.0001). Penicillin resistance was identified in 0.8% (95% CI 0%–4.2%) of isolates in the control group at 1 year, and in no isolates in the children-treated group at baseline or 1 year.

Conclusions

This cluster-randomized clinical trial demonstrated that compared to untreated control communities, nasopharyngeal pneumococcal resistance to macrolides was significantly higher in communities randomized to intensive azithromycin treatment. Mass azithromycin distributions were given more frequently than currently recommended by the World Health Organization''s trachoma program. Azithromycin use in this setting did not select for resistance to penicillins, which remain the drug of choice for pneumococcal infections.

Trial registration

www.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00322972","term_id":"NCT00322972"}}NCT00322972 Please see later in the article for the Editors'' Summary     

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP‐binding cassette transporter AdcCB, and, unusually, two zinc‐binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc‐specific substrate‐binding protein (SBP). By contrast with other zinc‐binding SBPs, AdcA has two zinc‐binding domains: a canonical amino‐terminal cluster A‐I zinc‐binding domain and a carboxy‐terminal zinc‐binding domain, which has homology to the zinc‐chaperone ZinT from Gram‐negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc‐binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.     

Short-sequence tandem and nontandem DNA repeats and endogenous hydrogen peroxide production contribute to genetic instability of Streptococcus pneumoniae

Loss-of-function mutations in the following seven pneumococcal genes were detected and analyzed: pspA, spxB, xba, licD2, lytA, nanA, and atpC. Factors associated with these mutations included (i) frameshifts caused by reversible gain and loss of single bases within homopolymeric repeats as short as 6 bases, (ii) deletions caused by recombinational events between nontandem direct repeats as short as 8 bases, and (iii) substitutions of guanine residues caused at an increased frequency by the high levels of hydrogen peroxide (>2 mM) typically generated by this species under aerobic growth conditions. The latter accounted for a frequency as high as 2.8 x 10(-6) for spontaneous mutation to resistance to optochin and was 10- to 200-fold lower in the absence of detectable levels of H2O2. Some of these mutations appear to have been selected for in vivo during pneumococcal infection, perhaps as a consequence of immune pressure or oxidative stress.     

Antibiotic sensitivity of Streptococcus pneumoniae strains isolated from the bronchial contents of patients with inflammatory lung diseases

The study of the sensitivity of 2681 pneumococcal strains to antibiotics, carried out in 1976-1982, demonstrated that most of these strains circulating in Leningrad were sensitive to penicillin, levomycetin, erythromycin and oleandomycin. Penicillin-resistant pneumococcal strains were found to appear in 1980 and later showed a definite increase in number, reaching 4.4% of all strains in 1982. Simultaneously the appearance of levomycetin- and erythromycin-resistant strains and the increase of their relative significance were established. Most of the pneumococcal strains isolated in Leningrad were not sensitive to monomycin, streptomycin and tetracyclin.     

Schistosomiasis may contribute to goblet cell carcinoid of the appendix

Abstract : To investigate whether schistosomiasis can contribute to appendiceal goblet cell carcinoid, appendix samples were obtained from 3 patients with combined appendiceal schistosomiasis and goblet cell carcinoid (CSG), 6 patients with goblet cell carcinoid only (GCC), 12 patients with appendiceal schistosomiasis only (ASO), and 12 cases with normal appendix (NA), all of similar gender ratio and age distributions. Hematoxylin and eosin-(H&E) stained sections were studied in 3 CSGs and 12 ASOs to diagnose schistosomiasis by detecting schistosome eggs. H&E and alcian blue/PAS-stained sections and immunohistochemistry of CgA and CEA were employed to establish the diagnosis of GCC in the 3 CSGs and 6 GCCs. Then, to determine whether schistosomiasis can contribute to GCC, immunostaining patterns of CgA and Ki67 in mucosal crypt epithelia were investigated and compared among all 33 cases. Our results revealed typical histological and immunohistochemical phenotypes of GCC in the 3 CSGs and 6 GCCs and schistosome egg deposits in 3 CSGs and 12 ASOs. We found that the expression levels of both CgA and Ki67 in mucosal crypt epithelia were significantly higher in CSG than in GCC (P < 0.05 = 0.013 and P = 0.004, respectively). Moreover, high expression levels of both CgA and Ki67 in mucosal crypt epithelia favor ASO as compared to NA (P < 0.001 = 3.4 × 10(-6) and 3.1 × 10(-5), respectively). Our findings suggest that appendiceal schistosomiasis was associated with increased proliferation and neuroendocrine differentiation of mucosal pluripotent crypt cells and that it may contribute to GCC, which is documented to originate from mucosal pluripotent crypt cells in mucosal crypt epithelia.     

Serotype Distribution,Antibiotic Resistance and Clonality of Streptococcus pneumoniae Isolated from Immunocompromised Patients in Tunisia

BackgroundPneumococcal disease, a major cause of morbidity and mortality globally, has higher incidence among young children, the elderly and the immunocompromised of all ages. In Tunisia, pneumococcal conjugate vaccines (PCVs) are not included in the national immunization program. Also, few studies have described the epidemiology of S. pneumoniae in this country and, in particular, no molecular typing studies have been performed. The aim of this study was to evaluate serotype distribution, antimicrobial resistance and clonality of Streptococcus pneumoniae isolated from neutropenic patients in Tunisia.MethodsFifty-nine S. pneumoniae were isolated from infection (n = 31) and colonization (n = 28) sites of patients (children and adults) attending the National Centre of Bone Marrow Transplantation in Tunis between 2005–2011. All isolates were characterized by serotype, antimicrobial resistance pattern and multilocus sequence typing (MLST).ResultsThe majority (66.1%) of the isolates belonged to five serotypes all included in PCVs: 6B, 9V, 14, 19F and 23F. The potential coverage of the 10-valent and 13-valent PCV was of 71.2% and 76.3% respectively. Resistance rates were very high and 69.5% of the isolates were multidrug resistant: non-susceptibility rates to penicillin, amoxicillin and cefotaxime were 66.1%, 40.7% and 27.1%, respectively; resistance rates to erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole, were 69.5%, 61.0%, 37.3%, 22.0% and 67.8%, respectively. The most frequent serotypes had STs characteristic of multidrug resistant international clones known to be highly successful and important causes of pneumococcal infection: Spain 23F-ST81, France 9V/14-ST156, Spain 6B-ST90, 19F-ST320, and Portugal 19F-ST177.ConclusionsThe majority of S. pneumoniae strains recovered from immunocompromised patients in Tunisia are representatives of multidrug resistant pandemic clones that express serotypes targeted by PCVs. To contain the burden of pneumococcal disease and improve treatment choices among Tunisian immunocompromised patients PCVs should be offered to all of them.     

Postprandial recruitment of neutrophils may contribute to endothelial dysfunction

Atherosclerosis is a low-grade inflammatory disease involving leukocytes, lipids, and glucose leading to endothelial dysfunction. Since activation of neutrophils by triglycerides and glucose has been described in vitro, we hypothesized that the postprandial phase is an inflammatory state affecting leukocytes, possibly contributing to endothelial dysfunction. We measured postprandial blood leukocyte counts, cytokines, hydroperoxides (HPOs), and flow-mediated vasodilation (FMD) in eight healthy males (age 23 +/- 2 years) after a FAT (50 g/m2) and GLUCOSE challenge (37.5 g/m2), a combination of both (MIXED test), and after WATER. All tests, except WATER, resulted in significantly impaired FMD (10% reduction) between t = 1 h and t = 3 h, accompanied by a significant increase of neutrophils (59% after FAT and 28% after GLUCOSE and MIXED), total plasma HPOs (15 to 31% increase), and plasma interleukin-8 (IL-8) (50-130% increase). WATER did not affect FMD, neutrophils, HPOs, or IL-8. Lymphocytes increased gradually in all tests (40-70% increase at t = 10 h compared with t = 0; P < 0.005), paralleling a gradual 3- to 5-fold interleukin-6 increase. Monocyte and erythrocyte counts did not change in any test. In conclusion, the neutrophil increment during postprandial lipemia and glycemia with concomitant IL-8 and HPO increases may contribute to endothelial dysfunction. Lymphocyte increment is a nonspecific diurnal process. Postprandial intravascular inflammatory changes may be relevant for the pathogenesis of atherosclerosis.     

Molecular mimicry may contribute to pathogenesis of ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with mucosal inflammation and ulceration of the colon. There seems to be no single etiological factor responsible for the onset of the disease. Autoimmunity has been emphasized in the pathogenesis of UC. Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) are common in UC, and recently two major species of proteins immunoreactive to pANCA were detected in bacteria from the anaerobic libraries. This implicates colonic bacterial protein as a possible trigger for the disease-associated immune response. Autoantibodies and T-cell response against human tropomyosin isoform 5 (hTM5), an isoform predominantly expressed in colon epithelial cells, were demonstrated in patients with UC but not in Crohn's colitis. We identified two bacterial protein sequences in NCBI database that have regions of significant sequence homology with hTM5. Our hypothesis is that molecular mimicry may be responsible for the pathogenesis of UC.     

Multiple mechanisms may contribute to the adherence ofCandida yeasts to living cells

The adherence ofCandida yeasts to monolayers of human intestinal epithelium was studied in order to determine the specific and nonspecific mechanisms that might contribute to yeast adherence. Multiple factors were shown to significantly affect the adherence of yeasts to intestinal cells. It was demonstrated that hydrophobic yeasts adhered two times greater than normal yeasts, and positively charged yeasts adhered ten times greater than normal yeasts to monolayers of intestinal epithelium. The binding of yeasts to the intestinal cells was saturable and was most effectively blocked by mucin, which caused an 83% reduction in adherence, whereas the addition ofd-glucose caused a 41% reduction in adherence. Aggregation or coadherence of yeasts occurred as the yeast inocula were increased.Candida appears to possess the ability to adhere to living tissue by several mechanisms, such as adhesin-receptor interactions, nonspecific hydrophobic and ionic bonding, and aggregation or coadherence. This is the first demonstration of multiple forces that may act simultaneously in the process of adherence of yeasts to living cells.     

MM1, a temperate bacteriophage of the type 23F Spanish/USA multiresistant epidemic clone of Streptococcus pneumoniae: structural analysis of the site-specific integration system

We have characterized a temperate phage (MM1) from a clinical isolate of the multiply antibiotic-resistant Spanish/American 23F Streptococcus pneumoniae clone (Spain(23F)-1 strain). The 40-kb double-stranded genome of MM1 has been isolated as a DNA-protein complex. The use of MM1 DNA as a probe revealed that the phage genome is integrated in the host chromosome. The host and phage attachment sites, attB and attP, respectively, have been determined. Nucleotide sequencing of the attachment sites identified a 15-bp core site (5'-TTATAATTCATCCGC-3') that has not been found in any bacterial genome described so far. Sequence information revealed the presence of an integrase gene (int), which represents the first identification of an integrase in the pneumococcal system. A 1.5-kb DNA fragment embracing attP and the int gene contained all of the genetic information needed for stable integration of a nonreplicative plasmid into the attB site of a pneumococcal strain. This vector will facilitate the introduction of foreign genes into the pneumococcal chromosome. Interestingly, DNAs highly similar to that of MM1 have been detected in several clinical pneumococcal isolates of different capsular types, suggesting a widespread distribution of these phages in relevant pathogenic strains.     

Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation

Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In Bacillus and Escherichia, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between Streptococcus pneumoniae recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in Bacillus but less than that in Escherichia. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from Bacillus, Escherichia, and Streptococcus.     

Collagen overglycosylation: a biochemical feature that may contribute to bone quality

Skeletal ability to resist mechanical stress is determined by bone amount and quality, which relies on macro- and micro-architecture, turnover, bone matrix, and mineralisation; the role of collagen has not been clearly elucidated. Numerous post-translational steps are involved in collagen type I biosynthesis, including residue hydroxylation and glycosylation catalysed by enzymes that work until the protein folds forming the triple helix; therefore, folding rate regulates these processes. Overglycosylated hydroxylysines are poor substrates for epsilon-amino group deamination which initiates cross-link formation. Three clinical conditions associated with fractures may relate collagen overglycosylation with bone quality: (i) Osteogenesis Imperfecta, in which genetic mutations distort triple helix conformation and slow folding rate favouring overglycosylation; (ii) diabetes mellitus, with collagen overglycosylation by AGE accumulation; and, (iii) menopause, according to experimental studies demonstrating ovariectomy-related trabecular bone collagen overglycosylation preventable by 17beta-estradiol or tamoxifen. Specific actions on collagen of drugs used for bone protection should be explored in future studies.     

The relBE2Spn Toxin-Antitoxin System of Streptococcus pneumoniae: Role in Antibiotic Tolerance and Functional Conservation in Clinical Isolates

Type II (proteic) chromosomal toxin-antitoxin systems (TAS) are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6). To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, ΔrelB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection.     

Identification of the optochin-resistant Streptococcus pneumoniae strains

So far the prevalent method of the identification of pneumococci in the routine diagnostic practice has included three tests: sensitivity to optoxin, solubility in biliary salts and reaction with omniserum. At present optoxin-resistant pneumococcal strains, as well as those giving dual reaction to solubility in biliary salts, occur sufficiently often thatconsiderably complicates the identification of this infective agent. We have summarized experience in the isolation of S. pneumoniae strains, resistant to optoxin and not soluble in biliary salts; in addition, the prospects of introducing the complex approach to the identification of a given culture with the use of molecular diagnostic methods into daily practice are evaluated.     

Serotype Distribution and Antibiotic Susceptibility of Streptococcus pneumoniae Strains Carried by Children Infected with Human Immunodeficiency Virus

Background

We studied the serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae isolates carried by children infected with HIV in Jakarta, Indonesia.

Methods

Nasopharyngeal swabs were collected from 90 HIV infected children aged 4 to 144 months. S. pneumoniae was identified by conventional and molecular methods. Serotyping was performed with sequential multiplex PCR and antibiotic susceptibility with the disk diffusion method.

Results

We identified S. pneumoniae carriage in 41 children (46%). Serotype 19F was most common among 42 cultured strains (19%) followed by 19A and 6A/B (10% each), and 23F (7%). Most isolates were susceptible to chloramphenicol (86%), followed by clindamycin (79%), erythromycin (76%), tetracycline (43%), and sulphamethoxazole/trimethoprim (41%). Resistance to penicillin was most common with only 33% of strains being susceptible. Strains of serotypes targeted by the 13-valent pneumococcal conjugate polysaccharide vaccine (PCV13) were more likely to be multidrug resistant (13 of 25 or 52%) compared to non-PCV13 serotype isolates (3 of 17 or 18%; Fisher exact test p = 0.05).

Conclusion

Our study provides insight into the epidemiology of pneumococcal carriage in young HIV patients in Indonesia. These findings may facilitate potential preventive strategies that target invasive pneumococcal disease in Indonesia.