Fluoroquinolone resistance in Streptococcus pneumoniae isolates in Germany from 2004-2005 to 2014-2015.

Streptococcus pneumoniae is a major cause of bacterial pneumonia, sepsis and meningitis worldwide. Prevalence of levofloxacin-resistant S. pneumoniae isolates in Germany and associated mutations in the quinolone resistance determining regions (QRDRs), as well as serotype distribution and multi locus sequence types (MLST) are shown. 21,764 invasive S. pneumoniae isolates from Germany, isolated in the epidemiological seasons from 2004/05 to 2014/15 were analyzed at the German National Reference Centre for Streptococci (GNRCS) for their levofloxacin resistance by micro broth dilution method. All resistant (minimal inhibitory concentration (MIC) ≥8µg/ml) and intermediate (MIC >2µg/ml and <8µg/ml) isolates were selected for the present study. Additionally, 29 susceptible isolates were randomly selected. A total of ninety isolates were tested for their levofloxacin-MIC by Etest, their serotype and sequence type, as well as for point-mutations at the QRDRs in the genes parC, parE, gyrA and gyrB. Twenty-five isolates exhibited levofloxacin MICs <2µg/ml (Etest) and no mutations in the QRDRs. Four isolates with MICs=2µg/ml had one mutation in parC; isolates with MICs >2µg/ml all had one or more mutations in the QRDRs. Four of nine intermediate isolates had a mutation in either parC or gyrA, and four isolates had mutations in both parC and gyrB. One isolate had mutations in both parC and gyrA. All isolates with MICs ≥8µg/ml (52) had mutations in both topoisomerase IV and gyrase. Serotypes associated with levofloxacin resistance shifted from a majority of PCV13 serotypes before the introduction of the PCV13 vaccine towards non-PCV serotypes. Resistant isolates were almost exclusively found among adults (98.1%).

Pneumococci in the African meningitis belt: meningitis incidence and carriage prevalence in children and adults.

BACKGROUND: The development of optimal vaccination strategies for pneumococcal conjugate vaccines requires serotype-specific data on disease incidence and carriage prevalence. This information is lacking for the African meningitis belt. METHODS: We conducted hospital-based surveillance of acute bacterial meningitis in an urban and rural population of Burkina Faso during 2007-09. Cerebrospinal fluid was evaluated by polymerase chain reaction for species and serotype. In 2008, nasopharyngeal swabs were obtained from a representative population sample (1 month to 39 years; N = 519) and additional oropharyngeal swabs from 145 participants. Swabs were evaluated by culture. RESULTS: Annual pneumococcal meningitis incidence rates were highest among <6-month-old (58/100,000) and 15- to 19-year-old persons (15/100,000). Annual serotype 1 incidence was around 5/100,000 in all age groups. Pneumococcal carriage prevalence in nasopharyngeal swabs was 63% among <5-year-old children and 22% among ≥5-year-old persons, but adding oropharyngeal to nasopharyngeal swabs increased the estimated carriage prevalence by 60%. Serotype 1 showed high propensity for invasive disease, particularly among persons aged ≥5 years. CONCLUSIONS: Serotype 1 causes the majority of cases with a relatively constant age-specific incidence. Pneumococcal carriage is common in all age groups including adults. Vaccination programs in this region may need to include older target age groups for optimal impact on disease burden.

Serotyping pneumococcal meningitis cases in the African meningitis belt by use of multiplex PCR with cerebrospinal fluid.

We reformulated a multiplex PCR algorithm for serotyping of pneumococcal meningitis directly on cerebrospinal fluid (CSF). Compared to established methods on isolates, CSF-based PCR had at least 80% sensitivity and 100% specificity. In regional meningitis surveillance, CSF-based PCR increased the serotype information yield from 40% of cases (isolate testing) to 90%.

Turgor regulation in the osmosensitive cut mutant of Neurospora crassa.

The internal hydrostatic pressure (turgor) of fungal cells is maintained at 400-500 kPa. The turgor is regulated by changes in ion flux and by production of the osmotically active metabolite glycerol. In Neurospora crassa, there are at least two genetically distinct pathways that function in adaptation to hyperosmotic shock. One involves a mitogen-activated protein (MAP) kinase cascade (kinases OS-4, OS-5 and OS-2 downstream of the osmosensing OS-1); the other is less understood, but involves the cut gene, which encodes a putative phosphatase. This study examined turgor regulation, electrical responses, ion fluxes and glycerol accumulation in the cut mutant. Turgor recovery after hyperosmotic treatment was similar to that in the wild-type, for both time-course ( approximately 40 min) and magnitude. Prior to turgor recovery, the hyperosmotic shock caused a rapid transient depolarization of the membrane potential, followed by a sustained hyperpolarization that occurred concomitant with increased H(+) efflux, indicating that the plasma membrane H(+)-ATPase was being activated. These changes also occurred in the wild-type. Net fluxes of Ca(2+) and Cl(-) during turgor recovery were similar to those in the wild-type, but K(+) influx was attenuated in the cut mutant. The similar turgor recovery can be explained by the ion uptake, since glycerol did not accumulate in the cut mutant within the time frame of turgor recovery (but did accumulate in the wild-type). The results suggest that turgor regulation involves multi-faceted coordination of both ion flux and glycerol accumulation. Ion uptake is activated by a MAP kinase cascade, while CUT is required for glycerol accumulation.

Role of a mitogen-activated protein kinase cascade in ion flux-mediated turgor regulation in fungi.

Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca(2+) influx and the sustained hyperpolarization is due to H(+) efflux by activation of the plasma membrane H(+)-ATPase. Protein synthesis is not required for H(+)-ATPase activation. Net K(+) and Cl(-) uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl(-) uptake increases, but net K(+) flux barely changes and net H(+) efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H(+)-ATPase, and net K(+) and Cl(-) uptake during turgor regulation. Other pathways regulating turgor must also exist.

The role of tip-localized mitochondria in hyphal growth.

Hyphal tip-growing organisms have a high density of tip-localized mitochondria which maintain a membrane potential based on Rhodamine 123 fluorescence, but do not produce ATP based on the absence of significant oxygen consumption. Two possible roles of these mitochondria in tip growth were examined: Calcium sequestration and biogenesis, because tip-high cytoplasmic calcium gradients are a common feature of tip-growing organisms, and the volume expansion as the tip extends would require a continuous supply of additional mitochondria. Co-localization of calcium-sensitive fluorescent dye and mitochondria-specific fluorescent dyes showed that the tip-localized mitochondria do contain calcium, and therefore, may function in calcium clearance from the cytoplasm. Short-term inhibition of DNA synthesis or mitochondrial protein synthesis did not affect either tip growth, or mitochondrial shape or distribution. Therefore, mitochondrial biogenesis may not occur from the tip-localized mitochondria in hyphal organisms.

Turgor regulation in hyphal organisms.

Turgor regulation in two saprophytic hyphal organisms was examined directly with the pressure probe technique. The ascomycete Neurospora crassa, a terrestrial fungi, regulates turgor after hyperosmotic treatments when growing in a minimal medium containing K(+), Mg(2+), Ca(2+), Cl(-), and sucrose. Turgor recovery by N. crassa after hyperosmotic treatment is concurrent with changes in ion transport: hyperpolarization of the plasma membrane potential and a decline in transmembrane ion conductance. In contrast the oomycete Achlya bisexualis, a freshwater hyphal organism, does not regulate turgor after hyperosmotic treatment, although small transient increases in turgor were occasionally observed. We also monitored turgor in both organisms during hypoosmotic treatment and did not observe a turgor increase, possibly due to turgor regulation. Both hyphal organisms grow with similar morphologies, cellular expansion rates and turgor (0.4-0.7 MPa), yet respond differently to osmotic stress. The results do not support the assumption of a universal mechanism of tip growth driven by cell turgor.

Oxygen flux magnitude and location along growing hyphae of Neurospora crassa.

Oxygen fluxes were mapped at the growing apices and along mycelial hyphal segments of the ascomycete Neurospora crassa. High spatial resolution was obtained using micro-oxygen probes (2-3 microm tip diameters) and the self-referencing technique to maximize the sensitivity of oxygen flux measurements. As expected, oxygen influx was inhibited by cyanide, although oxygen influx (and hyphal growth) resumed with the induction of an alternate oxidase activity. Along hyphal segments, variations in oxygen influx were not correlated with location, near or far from septa, and varied over time along the same hyphal segment. Growing hyphae had a region of maximal oxygen influx greater than 10 microm behind the hyphal tip, the oxygen influx was correlated with hyphal growth rate. The region of maximal oxygen influx did not correspond with mitochondrial density, which is maximal (about 30% of hyphal volume) 5-10 microm behind the tip. Therefore, tip-localized mitochondria do not contribute to the respiratory requirements of the growing hypha. The tip-localized mitochondria may function in clearing calcium from the cytoplasm, although a decline in chlortetracycline fluorescence after cyanide inhibition could also be due to ATP-depletion due to inhibition of actively respiring mitochondria. Alternatively, the growing tip may be the site of mitochondrial biogenesis.

Two divergent plasma membrane syntaxin-like SNAREs, nsyn1 and nsyn2, contribute to hyphal tip growth and other developmental processes in Neurospora crassa.

Highly polarized exocytosis of vesicles at hyphal apices is an essential requirement of tip growth. This requirement may be met by the localization and/or activation of an apical SNARE-based machinery. We have cloned nsyn1 and nsyn2, SNAREs predicted to function at the plasma membrane in Neurospora crassa. Transformation of extra copies of nsyn1 into wild-type strains displayed effects consistent with quelling of nsyn1 expression, which was lethal in most transformants. All surviving transformants grew slowly, conidiated poorly, and were male sterile. In addition, antisense nsyn1 strains grew slowly, with abnormal hyphal diameters and polarity and defective conidiation. For nsyn2, several repeat induced point mutation (RIP) crosses produced no, or poorly germinating ascospores. Those that germinated produced slow-growing hyphae with abnormal branching. The defects in nsyn1 and nsyn2 mutants are consistent with differential impaired vesicle fusion in hyphal tips and other developmental stages.