Evaluating the Rapid Emergence of Daptomycin Resistance in Corynebacterium: A Multi-Center Study

Background: Members of the genus Corynebacterium are increasingly recognized as pathobionts and can be very resistant to antimicrobial agents. Previous studies have demonstrated that Corynebacterium striatum can rapidly develop high-level daptomycin resistance (HLDR) (minimum inhibitory concentration [MIC] ≥256 μg/mL). Here we conducted a multi-center study to assay for this in vitro phenotype in diverse Corynebacterium species. Methods: Corynebacterium clinical isolates (n=157) from four medical centers were evaluated. MIC values to daptomycin, vancomycin, and telavancin were determined before and after overnight exposure to daptomycin to identify isolates able to rapidly develop daptomycin non-susceptibility. To investigate assay reproducibility, 18 isolates were evaluated at three study sites. In addition, stability of daptomycin non-susceptibility was tested using repeated subculture without selective pressure. The impact of different media brands was also investigated. Results: Daptomycin non-susceptibility emerged in 12 of 23 species evaluated in this study (C. afermentans, amycolatum, aurimucosum, bovis, jeikeium, macginleyi, pseudodiphtheriticum, resistens, simulans, striatum, tuberculostearicum, and ulcerans) and was detected in 50 of 157 (31.8%) isolates tested. All isolates displayed low (susceptible) MIC values to vancomycin and telavancin before and after daptomycin exposure. Repeated subculture demonstrated 2 of 9 isolates (22.2%) exhibiting HLDR reverted to a susceptible phenotype. Of 30 isolates tested on three media brands, 13 (43.3%) had differences in daptomycin MIC values between brands. Conclusions: Multiple Corynebacterium species can rapidly develop daptomycin non-susceptibility, including HLDR, after a short daptomycin exposure period.

Pengaruh jumlah subkultur dan media sub-optimal terhadap pertumbuhan dan kemampuan regenerasi kalus tebu (Saccharum officinarum L.) (Effect of Repeated Subculture and suboptimum media on the Growth of Sugarcane Calli (Saccharum officinarum L.))

Sugarcane (Saccharum officinarum L.) is an important crop for sugar production. One attempt to increase sugarcane productivity is through micropropagation and quality improvement of sugarcane seedlings in vitro. This research aimed to study the effect of repeated subcultures on callus capacity for regeneration and plant survival in acclimatization phase, as well as the influence of suboptimum media on the recovery capability of sugarcane callus to proliferate in vitro. Fourth subcultured sugarcane callus derived from young leaves were used as material in this research. Basic medium of Murashige and Skoog (MS) added with 3 mg/L 2,4-D, 10% coconut water, and 3% sucrose was used for callus initiation. For callus regeneration, the MS medium was supplemented with 2 mg/L BAP, 0.2 mg/L IAA, 10% coconut water, and 3% sucrose. Study on the effect of subculture numbers consisted of three stages, i.e. initiation, regeneration, and acclimatization, while the study on resting phase or the use of sub-optimal media included six treatment media and two pathways. Results showed that the fifth subcultures produced embryoid callus (91%), the highest non mucilaginous callus (97%), and the highest abnormality rate (6%). Results from the suboptimum media treatment, showed that B pathway (4 week resting phase) was better than the A pathway (8 week resting phase), based on fresh weight and callus abnormality percentage. A and B pathways indicated that the growth of callus can be recovered when it was grown back to the normal media and 1.5D-MS treatment of the resting phase showed the best growth and appearance.

Pengaruh jumlah subkultur dan media sub-optimal terhadap pertumbuhan dan kemampuan regenerasi kalus tebu (Saccharum officinarum L.) (Effect of repeated subculture and suboptimum media on the growth of sugarcane calli (Saccharum officinarum L.))

Sugarcane (Saccharum officinarum L.) is an important crop for sugar production. One attempt to increase sugarcane productivity is through micropropagation and quality improvement of sugarcane seedlings in vitro. This research aimed to study the effect of repeated subcultures on callus capacity for regeneration and plant survival in acclimatization phase, as well as the influence of suboptimum media on the recovery capability of sugarcane callus to proliferate in vitro. Fourth subcultured sugarcane callus derived from young leaves were used as material in this research. Basic medium of Murashige and Skoog (MS) added with 3 mg/L 2,4-D, 10% coconut water, and 3% sucrose was used for callus initiation. For callus regeneration, the MS medium was supplemented with 2 mg/L BAP, 0.2 mg/L IAA, 10% coconut water, and 3% sucrose. Study on the effect of subculture numbers consisted of three stages, i.e. initiation, regeneration, and acclimatization, while the study on resting phase or the use of sub-optimal media included six treatment media and two pathways. Results showed that the fifth subcultures produced embryoid callus (91%), the highest non mucilaginous callus (97%), and the highest abnormality rate (6%). Results from the suboptimum media treatment, showed that B pathway (4 week resting phase) was better than the A pathway (8 week resting phase), based on fresh weight and callus abnormality percentage. A and B pathways indicated that the growth of callus can be recovered when it was grown back to the normal media and 1.5D-MS treatment of the resting phase showed the best growth and appearance.

Clonal Propagation of Rhynchostylis retusa (Lin.) Blume through in vitro Culture and their Establishment in the Nursery

For high frequency regeneration of Rhyncnostylis retusa (Lin.) Blume apical nodal segments were used. Half strength MS + 2% sucrose + 1.5 mg/l BA + 0.5 mg/l NAA + 2 g/l peptone + 10% (v/v) coconut water (CW) + 0.5 g/l activated charcoal (AC) was the best nutrient medium, on which 89% cultures induced 8 microshoots per culture. Subculture of microshoots for further 8 weeks on the same nutrient medium enhanced the number of microshoots up to 95. For further proliferation of microshoots, their development into shoots as well as formation of secondary microshoots from the base of the old ones, the best medium was half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 150 mg/l L-glutamine. Plantlets with roots were obtained in half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 5.0 g/l banana powder, on which cent per cent shoots rooted within eight weeks. The pH of all the categories of cultures were maintained at 5.6 before adding 2.2 g/l gelrite and autoclaving, and the cultures were incubated at 2000 - 3000 lux for 16/8 hrs light/dark at 24 ± 2ºC. Regeneration of plantlets continued due to repeated subculture of microshoots and regenerants were acclimatized and established in the nursery. Plant Tissue Cult. & Biotech. 22(1): 1-11, 2012 (June) DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11242

Distribution and incidence of necrotic and non-necrotic strains of bean yellow mosaic virus in wild and crop lupins

A new strain of bean yellow mosaic virus (BYMV), a non-necrotic strain, was found in south-west Western Australia. It differs from the original necrotic strain of BYMV in that it does not kill Lupinus angustifolius (narrow-leafed lupin) plants, but causes symptoms of mottle and stunting, or dead growing points, fleshy expanded leaves, and stunting. A survey of L. angustifolius crops during September and October 1997 compared the distribution and incidence of the necrotic strain with that of the non-necrotic strain. Based on 1000 plants inspected at the edge of each crop, the necrotic strain was found in 100 of 102 crops while the non-necrotic strain was found in 64 of them. Incidences ranged from 0.3 to 56% (necrotic strain) and 0.1 to 7% (non-necrotic strain) of plants counted. Both strains were present over the whole range of the survey. Wild L. angustifolius and L. luteus (yellow lupin) populations were also inspected. The necrotic and non-necrotic strains were found in 31 and 9 of the 34 L. angustifolius populations examined, respectively. Incidences ranged from 0.1 to 28% (necrotic strain) and 0.1 to 3% (non-necrotic strain) of plants counted. BYMV was found in 9 of 11 wild L. luteus populations with incidences ranging from 0.3 to 7% of plants counted. In a separate survey in which samples of L. angustifolius crops, with necrotic symptoms suspected of being caused by Colletotrichum gloeosporioides (lupin anthracnose disease), were examined, 37 of 130 samples had typical necrotic BYMV symptoms. Samples with these necrotic symptoms also came from northern and eastern wheatbelt areas not normally associated with BYMV infection. When 8 BYMV isolates cultured by sap inoculation in Trifolium subterraneum (subterranean clover) were tested by aphid transmission to L. angustifolius plants in 1994 and again in 1997, the isolates of the 2 strains behaved the same on both occasions causing only necrotic (3 isolates) or non-necrotic (5 isolates) symptoms. Thus, despite repeated subculture by sap inoculation over a 3.5-year period, the 2 BYMV strains still remained distinct. An isolate collected from wild L. luteus in 1997 produced only non-necrotic symptoms in L. angustifolius. The non-necrotic strain caused symptoms typical of BYMV in hosts other than L. angustifolius, reacted strongly with BYMV antiserum, and failed to react with antiserum to clover yellow vein virus. In a BYMV-infected lupin crop, grain yields of individual L. angustifolius plants infected early with the non-necrotic strain were decreased by 95%. Shoot weights, seed number, and seed size were also greatly decreased. Widespread occurrence of the non-necrotic strain of BYMV is cause for concern for the lupin industry.

In Vitro Activities of Oral β-Lactams at Concentrations Achieved in Humans against Penicillin-Susceptible and -Resistant Pneumococci and Potential to Select Resistance

ABSTRACT The β-lactam susceptibilities of 65 strains ofStreptococcus pneumoniae for which penicillin MICs covered a broad range were assessed. The order of potency was amoxicillin (AMX) = amoxicillin-clavulanate (AMC) > penicillin G > cefpodoxime (CPO) > cefuroxime (CXM) > cefprozil > cefaclor > loracarbef > cefixime. No decrease in susceptibility was seen following repeated subculture of two penicillin-susceptible strains of S. pneumoniae in AMX, AMC, cefaclor, or loracarbef, whereas repeated exposure to CPO and CXM resulted in 4- to 32-fold decreases in susceptibility for both strains. When one of these strains was exposed to concentrations of CPO, CXM, AMX, and AMC achieved in the serum of humans following the administration of an oral dose, all agents were rapidly bactericidal, with no decrease in susceptibility up to 72 h. This was consistent with antibiotic concentrations exceeding the MICs for 100% of the dosing interval. For a penicillin-resistant strain, MICs were exceeded for 29% of the 12-h dosing interval for 500 mg of AMX, 42% of the interval for AMC with 875 mg of AMX and 125 mg of clavulanate (875/125 mg of AMC) 21% of the interval for 500 mg of CXM, and 0% of the interval for 200 mg of CPO. Consequently, only 875/125 mg of AMC produced a sustained bactericidal effect. A four- to eightfold reduction in susceptibility to CPO and CXM and cross-resistance with cefotaxime, but not penicillin or AMC, were selected following exposure to simulated serum CPO and CXM concentrations. In addition, AMX and AMC were the only agents which consistently produced a >99% reduction in bacterial numbers in time-kill studies using concentrations of antibiotic achieved in middle ear fluid for all three strains of penicillin-resistant S. pneumoniae tested.