Dried whole-plant Artemisia annua slows evolution of malaria drug resistance and overcomes resistance to artemisinin

Pharmaceutical monotherapies against human malaria have proven effective, although ephemeral, owing to the inevitable evolution of resistant parasites. Resistance to two or more drugs delivered in combination will evolve more slowly; hence combination therapies have become the preferred norm in the fight against malaria. At the forefront of these efforts has been the promotion of Artemisinin Combination Therapy, but despite these efforts, resistance to artemisinin has begun to emerge. In 2012, we demonstrated the efficacy of the whole plant (WP)—not a tea, not an infusion—as a malaria therapy and found it to be more effective than a comparable dose of pure artemisinin in a rodent malaria model. Here we show that WP overcomes existing resistance to pure artemisinin in the rodent malaria Plasmodium yoelii. Moreover, in a long-term artificial selection for resistance in Plasmodium chabaudi, we tested resilience of WP against drug resistance in comparison with pure artemisinin (AN). Stable resistance to WP was achieved three times more slowly than stable resistance to AN. WP treatment proved even more resilient than the double dose of AN. The resilience of WP may be attributable to the evolutionary refinement of the plant’s secondary metabolic products into a redundant, multicomponent defense system. Efficacy and resilience of WP treatment against rodent malaria provides compelling reasons to further explore the role of nonpharmaceutical forms of AN to treat human malaria.The fight against malaria predates the discovery of its causative agent, and for centuries malaria-associated fever was treated using herbal remedies. In the West, quinine (Cinchona bark extract) was the only affordable treatment against malaria until Paul Ehrlich’s magic bullet concept was adopted and thousands of synthetic compounds were tested against malaria parasites. Very few of these compounds were effective and/or safe for human use, but in the 1930s chloroquine rose to ascendancy as a miracle cure for malaria (1). In the late 1950s, chloroquine was the main weapon used by the World Health Organization (WHO) in its Global Malaria Eradication Program (GMEP). Sadly, development of drug-resistant parasites and concomitant failure of chloroquine as the drug of choice led to the eventual demise of GMEP by the close of the 1960s. Following chloroquine’s failure, various antimalarial compounds were serially deployed, and each in its turn failed as parasites evolved resistance, thus leaving millions of malaria patients without affordable treatment.In the 1970s, artemisinin was discovered as a pure drug extracted from the plant Artemisia annua. In wide-scale clinical trials, pure artemisinin showed poor pharmacokinetic properties but nonetheless demonstrated potent antimalarial activity with a high safety profile (2). It was determined that artemisinin when modified to artesunate or artemether improved bioavailability and was more effective when used in combination with other antimalarial drugs, mainly mefloquine, which became known as Artemisinin Combination Therapy (ACT) (3). It was hoped that use of ACTs would minimize risk of drug resistance. However, in 2005 the earliest evidence of P. falciparum resistance to ACTs arose in Southeast Asia (4–8). The fight against malaria became critical once again when it became apparent that ACT might be following chloroquine’s path toward obsolescence with no affordable replacement in sight.We demonstrated the efficacy of the whole A. annua plant as a malaria therapy and found it to be more effective than a comparable dose of pure artemisinin in a rodent malaria model (9). WHO has cautioned against use of nonpharmaceutical sources of artemisinin because of the risk of delivering subtherapeutic doses that could exacerbate the resistance problem (10). This warning is valid given the low artemisinin content of juice extractions, teas, and infusion preparations of plant material used for most nonpharmaceutical plant-based therapies. However, the Whole Plant (WP) A. annua therapy that we have tested is not an extraction, a tea, or an infusion, but is based on oral consumption of the dried leaves of the whole plant. Based on our proof of principle in a rodent model, we postulate that with further development WP might provide a more abundant and affordable source of artemisinin-based therapy by eliminating the need for artemisinin extraction during manufacture.WP may be more effective than monotherapeutic artemisinin because WP may constitute a naturally occurring combination therapy that augments artemisinin delivery and synergizes the drug’s activity. This plant Artemisinin Combination Therapy (pACT) is the result of evolutionary refinement of the plant’s secondary metabolic products into a resilient and multicomponent defense system. As was demonstrated for other combination therapies, we hypothesized that a WP-based pACT would (i) overcome existing resistance to monotherapeutic pure artemisinin and (ii) increase the longevity of this therapy by delaying the onset of parasite resistance among wild types. Here, we tested these two hypotheses in two mouse malaria models, artemisinin-resistant Plasmodium yoelii (strain: ART) and artemisinin-sensitive Plasmodium chabaudi (strain: ASS).     

From the Cover: Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes

The marine cyanobacterium Prochlorococcus is the most abundant photosynthetic organism in oligotrophic regions of the oceans. The inability to assimilate nitrate is considered an important factor underlying the distribution of Prochlorococcus, and thought to explain, in part, low abundance of Prochlorococcus in coastal, temperate, and upwelling zones. Here, we describe the widespread occurrence of a genomic island containing nitrite and nitrate assimilation genes in uncultured Prochlorococcus cells from marine surface waters. These genes are characterized by low GC content, form a separate phylogenetic clade most closely related to marine Synechococcus, and are located in a different genomic region compared with an orthologous cluster found in marine Synechococcus strains. This sequence distinction suggests that these genes were not transferred recently from Synechococcus. We demonstrate that the nitrogen assimilation genes encode functional proteins and are expressed in the ocean. Also, we find that their relative occurrence is higher in the Caribbean Sea and Indian Ocean compared with the Sargasso Sea and Eastern Pacific Ocean, which may be related to the nitrogen availability in each region. Our data suggest that the ability to assimilate nitrite and nitrate is associated with microdiverse lineages within high- and low-light (LL) adapted Prochlorococcus ecotypes. It challenges 2 long-held assumptions that (i) Prochlorococcus cannot assimilate nitrate, and (ii) only LL adapted ecotypes can use nitrite. The potential for previously unrecognized productivity by Prochlorococcus in the presence of oxidized nitrogen species has implications for understanding the biogeography of Prochlorococcus and its role in the oceanic carbon and nitrogen cycles.     

Antibiotics resistance rate of Helicobacter pylori in Bhutan

AIM:To survey the antibiotic resistance pattern of Helicobacter pylori(H.pylori)strains isolated from Bhutanese population.METHODS:We isolated 111 H.pylori strains from the gastric mucosa of H.pylori-infected patients in Bhutan in 2010.The Epsilometer test was used to determine the minimum inhibitory concentrations(MICs)of amoxicillin(AMX),clarithromycin(CLR),metronidazole(MNZ),levofloxacin(LVX),ciprofloxacin(CIP),and tetracycline(TET).RESULTS:Nineteen of the isolated H.pylori strains were susceptible to all antibiotics tested.The isolated strains showed the highest rate of antibiotic resistance to MNZ(92/111,82.9%).Among the 92 MNZresistant strains,74 strains(80.4%)showed high-level resistance(MIC≥256 g/mL).Three strains were resistance to LVX(2.7%).These strains were also resistance to CIP.None of the strains showed resistance to CLR,AMX and TET.CONCLUSION:CLR-based triple therapy is a more effective treatment approach over MNZ-based triple therapy for H.pylori infection in Bhutan.     

Drug interactions modulate the potential for evolution of resistance

Antimicrobial treatments increasingly rely on multidrug combinations, in part because of the emergence and spread of antibiotic resistance. The continued effectiveness of combination treatments depends crucially on the frequency with which multidrug resistance arises. Yet, it is unknown how this propensity for resistance depends on cross-resistance and on epistatic interactions—ranging from synergy to antagonism—between the drugs. Here, we analyzed how interactions between pairs of drugs affect the spontaneous emergence of resistance in the medically important pathogen Staphylococcus aureus. Resistance is selected for within a window of drug concentrations high enough to inhibit wild-type growth but low enough for some resistant mutants to grow. Introducing an experimental method for high-throughput colony imaging, we counted resistant colonies arising across a two-dimensional matrix of drug concentrations for each of three drug pairs. Our data show that these different drug combinations have significantly different impacts on the size of the window of drug concentrations where resistance is selected for. We framed these results in a mathematical model in which the frequencies of resistance to single drugs, cross-resistance, and epistasis combine to determine the propensity for multidrug resistance. The theory suggests that drug pairs which interact synergistically, preferred for their immediate efficacy, may in fact favor the future evolution of resistance. This framework reveals the central role of drug epistasis in the evolution of resistance and points to new strategies for combating the emergence of drug-resistant bacteria.     

A shift toward birthing relatively large infants early in human evolution

It has long been argued that modern human mothers give birth to proportionately larger babies than apes do. Data presented here from human and chimpanzee infant:mother dyads confirm this assertion: humans give birth to infants approximately 6% of their body mass, compared with approximately 3% for chimpanzees, even though the female body weights of the two species are moderately convergent. Carrying a relatively large infant both pre- and postnatally has important ramifications for birthing strategies, social systems, energetics, and locomotion. However, it is not clear when the shift to birthing large infants occurred over the course of human evolution. Here, known and often conserved relationships between adult brain mass, neonatal brain mass, and neonatal body mass in anthropoids are used to estimate birthweights of extinct hominid taxa. These estimates are resampled with direct measurements of fossil postcrania from female hominids, and also compared with estimates of female body mass to assess when human-like infant:mother mass ratios (IMMRs) evolved. The results of this study suggest that 4.4-Myr-old Ardipithecus possessed IMMRs similar to those found in African apes, indicating that a low IMMR is the primitive condition in hominids. Australopithecus females, in contrast, had significantly heavier infants compared with dimensions of the femoral head (n = 7) and ankle (n = 7) than what is found in chimpanzees, and are estimated to have birthed neonates more than 5% of their body mass. Carrying such proportionately large infants may have limited arboreality in Australopithecus females and may have selected for alloparenting behavior earlier in human evolution than previously thought.     

Mitochondrial heteroplasmy and the evolution of insecticide resistance: Non-Mendelian inheritance in action

Genes encoded by mitochondrial DNA (mtDNA) exist in large numbers per cell but can be selected very rapidly as a result of unequal partitioning of mtDNA between germ cells during embryogenesis. However, empirical studies of this “bottlenecking” effect are rare because of the apparent scarcity of heteroplasmic individuals possessing more than one mtDNA haplotype. Here, we report an example of insecticide resistance in an arthropod pest (Tetranychus urticae) being controlled by mtDNA and on its inheritance in a heteroplasmic mite strain. Resistance to the insecticide bifenazate is highly correlated with remarkable mutations in cytochrome b, a mitochondrially encoded protein in the respiratory pathway. Four sites in the Q_o site that are absolutely conserved across fungi, protozoa, plants, and animals are mutated in resistant mite strains. Despite the unusual nature of these mutations, resistant mites showed no fitness costs in the absence of insecticide. Partially resistant strains, consisting of heteroplasmic individuals, transmit their resistant and susceptible haplotypes to progeny in highly variable ratios consistent with a sampling bottleneck of ≈180 copies. Insecticide selection on heteroplasmic individuals favors those carrying resistant haplotypes at a frequency of 60% or more. This combination of factors enables very rapid evolution and accounts for mutations being fixed in most field-collected resistant strains. The results provide a rare insight into non-Mendelian mechanisms of mitochondrial inheritance and evolution, relevant to anticipating and understanding the development of other mitochondrially encoded adaptations in arthropods. They also provide strong evidence of cytochrome b being the target site for bifenazate in spider mites.     

维生素C注射液和头孢曲松钠用于复发性口腔溃疡的效果比较

[目的]观察维生素C和头孢曲松钠用于复发性口腔溃疡护理的效果。[方法]将2012年1月—2014年1月本科收治的复发性口腔溃疡病人90例,按方便抽样的方法随机分为两组。对照组给予维生素B2口服;观察组将1支国产头孢曲松钠和1支维生素C注射液调匀成糊状,置于玻璃容器内,用棉签蘸取少许涂于溃疡表面,每天3次,共治疗6d。比较两组治疗后效果、进食改善至正常的时间、口腔溃疡愈合时间。[结果]两组治疗后的效果、进食改善至正常的时间、口腔溃疡愈合时间比较差异有统计学意义(P<0.05)。[结论]采用维生素C和头孢曲松钠用于口腔溃疡效果好,无明显不良反应。     

Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.Poly(ADP-ribose) polymerase (PARP) inhibition provides a promising therapeutic strategy for targeting homologous recombination (HR)-deficient tumors, such as breast cancer 1 (BRCA1)-mutated cancers (1). Indeed, clinical phase I and phase II trials have shown potent anticancer activity of small molecule inhibitors of PARP, such as olaparib, in patients with BRCA1-associated breast cancer (2, 3). However, it remains to be established whether different breast cancer subtypes in BRCA1 mutation carriers respond equally to PARP inhibition. Reduced sensitivity of breast cancers to anticancer drugs has frequently been associated with an epithelial-to-mesenchymal transition (EMT) (4–7). Metaplastic breast carcinomas (MBCs) are a subset of triple-negative breast cancers (TNBCs) characterized by a claudin-low and EMT-like phenotype (8) and a poor prognosis compared with other TNBCs (9). More than 60% of MBCs have BRCA1 promoter methylation, raising the question whether these tumors can be effectively targeted by using PARP inhibitors (10). To address this issue in an experimentally controlled setting, we set out to generate a genetically engineered mouse model (GEMM) of BRCA1-deficient MBC by inducing EMT via MET overexpression in a previously established GEMM of BRCA1-mutated breast cancer. We report that EMT is associated with olaparib resistance and can be effectively bypassed by administration of AZD2461, a PARP inhibitor with low affinity for the P-glycoprotein (Pgp) drug efflux transporter.     

Suppression of P-gp induced multiple drug resistance in a drug resistant gastric cancer cell line by overexpression of Fas

AIM:To observe the drug sensitizing effect and related mechanisms of fas gene transduction on human drug-resistant gastric cancer cell SGC7901/VCR (resistant to Vincristine).METHODS:The cell cycle alteration was observed by FACS. The sensitivity of gastric cancer cells to apoptosis was determined by in vitro apoptosis assay. The drug sensitization of cells to several anti-tumor drugs was observed by MTT assay. Immunochemical method was used to show expression of P-gp and Topo II in gastric cancer cells.RESULTS:Comparing to SGC7901 and pBK-SGC7901/VCR, fas-SGC7901/VCR showed decreasing G2 cells and increasing S cells, the G2 phase fraction of pBK-SGC7901/VCR was about 3.0 times that of fas -SGC7901/VCR but S phase fraction of fas -SGC7901/VCR was about 1.9 times that of pBK-SGC7901/VCR, indicating S phase arrest of fas-SGC7901/VCR. FACS also suggested apoptosis of fas-SGC7901/VCR.fas-SGC7901/VCR was more sensitive to apoptosis inducing agent VM-26 than pBK-SGC7901/VCR. MTT assay showed increased sensitization of fas-SGC7901/VCR to DDP, MMC and 5-FU, but same sensitization to VCR according to pBK-SGC7901/VCR. SGC7901, PBK-SGC7901/VCR and fas -SGC7901/VCR had positively stained Topo II equally. P-gp staining in pBK-SGC7901/VCR was stronger than in SGC7901, but there was little staining of P-gp in fas-SGC7901/VCR.CONCLUSION:fas gene transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree, possibly because of higher sensitization to apoptosis and decreased expression of P-gp.     

Genomic analysis of diversity,population structure,virulence, and antimicrobial resistance in Klebsiella pneumoniae,an urgent threat to public health

Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.The Gram-negative bacterium Klebsiella pneumoniae is a leading cause of hospital-acquired (HA) infections and neonatal sepsis globally (1–3). Widely considered an opportunistic pathogen, K. pneumoniae can be carried asymptomatically in the intestinal tract, skin, nose, and throat of healthy individuals (4, 5) but can also cause a range of infections in hospitalized patients, most commonly pneumonia, wound, soft tissue, or urinary tract infections. K. pneumoniae infections are particularly a problem among neonates, the elderly, and the immunocompromised (4) but also cause significant numbers of serious community-acquired (CA) infections, including pyogenic liver abscess, pneumonia, and meningitis (6). Virulence factors thought to be associated with invasive CA infections include various siderophores, specific polysaccharide capsule serotypes, and rmpA genes that are associated with hypermucoidy (7).K. pneumoniae, particularly when hypermucoid, can cause invasive disease in several animal species (8, 9) and is a common cause of mastitis in dairy herds (10). Moreover it can thrive in a range of plant hosts and environmental niches, including water, soil, and plant matter (4, 5, 11). Although it is clear that K. pneumoniae is genetically and phenotypically diverse (12, 13), previous efforts to identify specific features that can distinguish human clinical isolates from plant, animal, or environmental isolates have yielded no markers of human-specific lineages (14). Three distinct phylogroups of K. pneumoniae—KpI, KpII, and KpIII—have been defined based on sequencing of a small number of genes (15, 16), and it has been proposed that these phylogroups be redesignated as distinct species, namely, K. pneumoniae (KpI), K. quasipneumoniae (KpII) (17), and K. variicola (KpIII) (18); however, all three cause infections in humans (15, 19).Critically, the emergence of multiple drug-resistant (MDR) K. pneumoniae has been identified as an urgent threat to human health, featuring, for example, in recent reports on antimicrobial resistance (AMR) from the US Centers for Disease Control and Prevention (CDC) (20) and the UK Department of Health (21), because of a high prevalence of resistance to carbapenems and broad-spectrum β-lactams (22–25). The most notorious example of AMR K. pneumoniae is a lineage identified as clonal complex (CC) 258 by multilocus sequence typing (MLST) (13); CC258 frequently carries the K. pneumoniae carbapenemase (KPC) gene as well as numerous other acquired AMR genes and has been responsible for hospital outbreaks on several continents (13, 26, 27).The tracking of AMR organisms is one of the four core actions proposed in the CDC AMR action plan to limit the emergence and spread of AMR bacteria. Several recent genomic analyses indicate that sequence type (ST) 258 is a recombinant strain that has undergone capsular exchange since its emergence as a cause of KPC outbreaks (28–30). However, little attention has been paid to other MDR clones, which also are common and can spread carbapenem resistance (31). Relatively little is known about this broader population of K. pneumoniae, and there remains a lack of data regarding transmission, pathogenicity, and the evolution and spread of MDR clones globally. Moreover, K. pneumoniae is considered a source and a reservoir of AMR genes, with many of the major families being described first in K. pneumoniae (22–25) before being identified in a range of other Gram-negative bacteria; hence it is crucial to improve our understanding of the broader population of K. pneumoniae beyond a handful of well-known clones. Many consider this knowledge to be fundamental to support efforts to control the threat to human health posed by this bacterium.With this aim, we sequenced the genomes of nearly 300 diverse K. pneumoniae isolates spanning four continents and collected from a range of human and animal sources, including infection, colonization, and the environment (Dataset S1). We also performed a pangenome-wide association study (PGWAS) to look for associations between gene repertoire and disease potential/outcome and to identify distinct sets of accessory genes associated with virulence traits in humans, world-wide.     

Antibiotic resistance and cagA gene correlation: a looming crisis of Helicobacter pylori

AIM: To determine antibiotic resistance of Helicobacter pylori (H. pylori) in Pakistan and its correlation with host and pathogen associated factors.METHODS: A total of 178 strains of H. pylori were isolated from gastric biopsies of dyspeptic patients. Susceptibility patterns against first and second-line antibiotics were determined and trends of resistance were analyzed in relation to the sampling period, gastric conditions and cagA gene carriage. The effect of cagA gene on the acquisition of resistance was investigated by mutant selection assay.RESULTS: The observations showed that monoresistant strains were prevalent with rates of 89% for metronidazole, 36% for clarithromycin, 37% for amoxicillin, 18.5% for ofloxacin and 12% for tetracycline. Furthermore, clarithromycin resistance was on the rise from 2005 to 2008 (32% vs 38%, P = 0.004) and it is significantly observed in non ulcerative dyspeptic patients compared to gastritis, gastric ulcer and duodenal ulcer cases (53% vs 20%, 18% and 19%, P = 0.000). On the contrary, metronidazole and ofloxacin resistance were more common in gastritis and gastric ulcer cases. Distribution analysis and frequencies of resistant mutants in vitro correlated with the absence of cagA gene with metronidazole and ofloxacin resistance.CONCLUSION: The study confirms the alarming levels of antibiotic resistance associated with the degree of gastric inflammation and cagA gene carriage in H. pylori strains.     

Stable isotope evidence for an amphibious phase in early proboscidean evolution

The order Proboscidea includes extant elephants and their extinct relatives and is closely related to the aquatic sirenians (manatees and dugongs) and terrestrial hyracoids (hyraxes). Some analyses of embryological, morphological, and paleontological data suggest that proboscideans and sirenians shared an aquatic or semiaquatic common ancestor, but independent tests of this hypothesis have proven elusive. Here we test the hypothesis of an aquatic ancestry for advanced proboscideans by measuring delta(18)O in tooth enamel of two late Eocene proboscidean genera, Barytherium and Moeritherium, which are sister taxa of Oligocene-to-Recent proboscideans. The combination of low delta(18)O values and low delta(18)O standard deviations in Barytherium and Moeritherium matches the isotopic pattern seen in aquatic and semiaquatic mammals, and differs from that of terrestrial mammals. delta(13)C values of these early proboscideans suggest that both genera are likely to have consumed freshwater plants, although a component of C(3) terrestrial vegetation cannot be ruled out. The simplest explanation for the combined evidence from isotopes, dental functional morphology, and depositional environments is that Barytherium and Moeritherium were at least semiaquatic and lived in freshwater swamp or riverine environments, where they grazed on freshwater vegetation. These results lend new support to the hypothesis that Oligocene-to-Recent proboscideans are derived from amphibious ancestors.     

Mechanisms of reduced susceptibility to ciprofloxacin in Escherichia coli isolates from Canadian hospitals

OBJECTIVE:

To determine whether plasmid-mediated quinolone resistance (PMQR) determinants play a role in the increasing resistance to fluoroquinolones among Escherichia coli isolates in Canadian hospitals, and to determine the mechanisms of reduced susceptibility to ciprofloxacin in a recent collection of 190 clinical E coli isolates.

METHODS:

E coli isolates (n=1702) were collected as part of the 2007 Canadian Hospital Ward Antibiotic Resistance Surveillance (CANWARD) study. Antimicrobial susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. Using a representative subset of isolates (n=190), the mechanisms of reduced susceptibility to ciprofloxacin were detected by polymerase chain reaction and sequencing of the quinolone resistance-determining regions (QRDR) of chromosomal gyrA and parC genes, and by polymerase chain reaction for the PMQR genes: qnr, aac(6′) Ib-cr and qepA.

RESULTS:

2.1% and 1.1% of E coli harboured aac(6′)Ib-cr and qnrB, respectively. Single amino acid substitutions in the QRDR of gyrA were observed among isolates with ciprofloxacin minimum inhibitory concentrations as low as 0.12 μg/mL. As the ciprofloxacin minimum inhibitory concentration increased to 1 μg/mL (which is still considered to be susceptible by the CLSI), the vast majority of isolates demonstrated both gyrA and parC mutations.

CONCLUSION:

PMQR determinants and QRDR mutants among clinical E coli isolates with reduced susceptibility to ciprofloxacin demonstrates the need for increased surveillance and the need to re-evaluate the current CLSI breakpoints to prevent further development of fluoroquinolone resistance.     

Penicillin susceptibility and macrolide-lincosamide-streptogramin B resistance in group B Streptococcus isolates from a Canadian hospital

BACKGROUND:

Intrapartum antibiotic prophylaxis (IAP) is recommended for pregnant women who test positive for group B Streptococcus (GBS) in their genitourinary tract to prevent GBS-induced neonatal sepsis. Penicillin G is used as the primary antibiotic, and clindamycin or erythromycin as the secondary, if allergies exist. Decreased susceptibility to penicillin G has occasionally been reported; however, clindamycin and erythromycin resistance is on the rise and is causing concern over the use of clindamycin and erythromycin IAP.

METHODS:

Antibiotic resistance was characterized phenotypically using a D-Test for erythromycin and clindamycin, while an E-Test (E-strip) was used for penicillin G. GBS was isolated from vaginal-rectal swabs and serologically confirmed using Prolex (Pro-Lab Diagnostics, Canada) streptococcal grouping reagents. Susceptibility testing of isolates was performed according to the Clinical Laboratory Standards Institute guidelines.

RESULTS:

All 158 isolates were penicillin G sensitive. Inducible macrolide-lincosamide-streptogramin B (MLSB) resistance was observed in 13.9% of isolates. Constitutive MLSB resistance was observed in 12.7% of isolates. M phenotype resistance was observed in 6.3% of isolates. In total, erythromycin resistance was present in 32.9% of the GBS isolates, while clindamycin resistance was present in 26.6%.

DISCUSSION:

The sampled GBS population showed no sign of reduced penicillin susceptibility, with all being well under susceptible minimum inhibitory concentration values. These data are congruent with the large body of evidence showing that penicillin G remains the most reliable clinical antibiotic for IAP. Clindamycin and erythromycin resistance was higher than expected, contributing to a growing body of evidence that suggests the re-evaluation of clindamycin and erythromycin IAP is warranted.     

Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains

The toxin-producing bacterium C. difficile is the leading cause of antibiotic-associated colitis, with an estimated 500,000 cases C. difficile infection (CDI) each year in the US with a cost approaching 3 billion dollars. Despite the significance of CDI, the pathogenesis of this infection is still being defined. The recent development of tractable murine models of CDI will help define the determinants of C. difficile pathogenesis in vivo. To determine if cefoperazone-treated mice could be utilized to reveal differential pathogenicity of C. difficile strains, 5-8 week old C57BL/6 mice were pretreated with a 10 d course of cefoperazone administered in the drinking water. Following a 2-d recovery period without antibiotics, the animals were orally challenged with C. difficile strains chosen to represent the potential range of virulence of this organism from rapidly fatal to nonpathogenic. Animals were monitored for loss of weight and clinical signs of colitis. At the time of harvest, C. difficile strains were isolated from cecal contents and the severity of colitis was determined by histopathologic examination of the cecum and colon. Cefoperazone treated mice challenged with C. difficile strains VPI 10463 and BI1 exhibited signs of severe colitis while infection with 630 and F200 was subclinical. This increased clinical severity was correlated with more severe histopathology with significantly more edema, inflammation and epithelial damage encountered in the colons of animals infected with VPI 10463 and BI1. Disease severity also correlated with levels of C. difficile cytotoxic activity in intestinal tissues and elevated blood neutrophil counts. Cefoperazone treated mice represent a useful model of C. difficile infection that will help us better understand the pathogenesis and virulence of this re-emerging pathogen.     

Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38

The baculovirus Cydia pomonella granulovirus (CpGV) is widely applied as a biocontrol agent of codling moth. After field resistance of codling moth populations had been observed against the commercially used Mexican (M) isolate of CpGV, infection experiments of larvae of the resistant codling moth strain CpRR1 showed that several other naturally occurring CpGV isolates (I12, S, E2, and I07) from different geographic origins are still infectious to resistant CpRR1. Whole-genome sequencing and phylogenetic analyses of these geographic CpGV variants revealed that their genomes share only a single common difference from that of CpGV-M, which is a mutation coding for a repeat of 24 nucleotides within the gene pe38; this mutation results in an additional repeat of eight amino acids that appears to be inserted to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abolished virus infection in codling moth cells and larvae, demonstrating that it is an essential gene. When the CpGV-M deletion mutant was repaired with pe38 from isolate CpGV-S, which originated from the commercial product Virosoft and is infectious for the resistant codling moth strain CpRR1, the repaired CpGV-M mutant was found to be fully infectious for CpRR1. Repair using pe38 from CpGV-M restored infectivity for the virus in sensitive codling moth strains, but not in CpRR1. Therefore, we conclude that CpGV resistance of codling moth is directed to CpGV-M but not to other virus isolates. The viral gene pe38 is not only essential for the infectivity of CpGV but it is also the key factor in overcoming CpGV resistance in codling moth.The codling moth Cydia pomonella L. is a worldwide occurring insect pest that infests apples, pears, and walnuts. The larvae of codling moth bore into the fruit and cause severe economic damage if not controlled. A number of chemical and biological agents are available for the control of codling moth. One of the most efficient biological control agents (1–5) is the Cydia pomonella granulovirus (CpGV), which belongs to the dsDNA virus family Baculoviridae (genus Betabaculovirus). CpGV was first discovered in Mexico (Mexican isolate, CpGV-M) in 1963 (6); this isolate was later developed to commercial products now registered in 34 countries worldwide. The genome sequence of the in vivo cloned strain CpGV-M1 is ∼123 kbp and encodes for 143 ORFs (7). Based on SNPs in highly conserved genes, different geographic CpGV isolates were classified into four genome types, A–D (8). CpGV exhibits an extremely narrow host range that is restricted to C. pomonella and a very few closely related tortricids (Lepidoptera) (9).The development of resistance to baculovirus infection was thought to be unlikely before 2005 (10, 11), which is when the first cases of resistant codling moth populations with a 1,000- to 100,000-fold reduced susceptibility to commercial CpGV products containing the isolate CpGV-M were reported from organic apple plantations in Germany and France, where CpGV products had been intensively applied (12, 13). Since then, 38 apple plantations with CpGV resistance have been identified in Austria (2 orchards), Czech Republic (1), France (3), Germany (22), Italy (6), Switzerland (2), and the Netherlands (2) (14). For the codling moth strain CpR, which originated from a resistant field population in south Germany, as well as the genetically homogenous laboratory strain CpRR1, which derived from CpR, the mode of inheritance was revealed to be incompletely dominant, monogenic, and linked to the Z (sex) chromosome (15, 16). A similar mode of inheritance was also observed in resistant Czech and French codling moth populations arguing for a more or less universal mode of resistance in Europe (17, 18). These data led to the hypothesis that a genetic adaptation of codling moth to CpGV-M infection had occurred and was selected for by the intensive use of products containing CpGV-M (15).Insects manifest miscellaneous strategies to resist pathogens but lack an adaptive immune system. Insect defense to viral infections involves nonspecific factors such as physical barriers, enzymatic responses, and increasing ejection of infected midgut cells as larvae age, as well as specific factors involving cellular and humoral immunity (19). Physical barriers to infection include the perithropic membrane (PM), an ultrafilter for particles with size exclusion greater than 30 nm (20). Melanization of the cuticula mediated by phenoloxidase enzymes is involved in the encapsulation reaction of pathogens, such as bacteria, fungi, or virus-infected cells (21, 22). Developmental resistance, the decreasing susceptibility of larvae with increasing age, is rather common, and is mediated by infected midgut cell-sloughing (23–25). Behavior modifications also count among insect defense strategies (19). Feeding behaviors, e.g., can strongly affect the risk of insects for baculovirus infection as shown for Lymantria dispar L., which exhibit heritable cadaver-avoidance behaviors (26).An alternative way for insects to get rid of virus-infected cells is via programmed cell death—the apoptosis pathway (27). Baculoviruses, however, are able to block apoptosis with the help of different virus encoded antiapoptotic proteins. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV), these proteins—namely P35 and P49—are inhibitors of the insect caspases. These inhibitors of apoptosis (IAPs), thereby, ensure a permissive virus infection (28).For codling moth, however, resistance based on the PM, the midgut, or the immune system has been excluded for CpGV (16, 29). Lack of CpGV DNA replication and a systemic resistance in all five instars indicated an early block to virus replication in resistant codling moth individuals (29, 30). At the same time, it was observed that certain CpGV isolates, such as I12 or NPP-R1/R4, were able to infect larvae from resistant codling moth strains (30, 31). Therefore, these naturally occurring CpGV isolates from different geographic origins were considered resistance-breaking isolates. Some of these isolates, meanwhile, replace CpGV-M in commercial biocontrol agents in Europe and demonstrate the importance of identifying resistance-breaking CpGV variants (30–32) for managing CpGV-resistant codling moth populations; however, their functional difference to CpGV-M allowing them to overcome CpGV resistance remained unknown.In this study we compared different naturally occurring geographic CpGV isolates representing all known CpGV genome types (8) in laboratory assays for their infectivity to susceptible (CpS) and resistant (CpRR1) codling moth strains. We found that all tested isolates—except CpGV-M—were able to overcome resistance in CpRR1 larvae. Whole-genome sequencing of these isolates revealed a single common difference in all resistance-breaking isolates, which was located in ORF24 (pe38). Therefore, occlusion bodies of bacmid-based recombinant CpGV (in the following termed pseudoviruses) knockout and recovery mutants of pe38 were established and tested for their activity in susceptible CpS and resistant CpRR1 larvae. The recovery of infectivity of a CpGV-M–based pseudovirus harboring the pe38 of the resistance-breaking CpGV-S in CpRR1 larvae demonstrated the key function of pe38 in overcoming baculovirus resistance in codling moth. In their entirety, these investigations demonstrate the significance of using multiple isolates of CpGV concurrently in field applications to avoid resistance in codling moth populations.     

Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems

Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gram-negative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn²⁺ alone. The crystal structure of ZneB to 2.8 Å resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the β-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.     

Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis

Neisseria meningitidis is a leading cause of infectious childhood mortality worldwide. Most research efforts have hitherto focused on disease isolates belonging to only a few hypervirulent clonal lineages. However, up to 10% of the healthy human population is temporarily colonized by genetically diverse strains mostly with little or no pathogenic potential. Currently, little is known about the biology of carriage strains and their evolutionary relationship with disease isolates. The expression of a polysaccharide capsule is the only trait that has been convincingly linked to the pathogenic potential of N. meningitidis. To gain insight into the evolution of virulence traits in this species, whole-genome sequences of three meningococcal carriage isolates were obtained. Gene content comparisons with the available genome sequences from three disease isolates indicate that there is no core pathogenome in N. meningitidis. A comparison of the chromosome structure suggests that a filamentous prophage has mediated large chromosomal rearrangements and the translocation of some candidate virulence genes. Interspecific comparison of the available Neisseria genome sequences and dot blot hybridizations further indicate that the insertion sequence IS1655 is restricted only to N. meningitidis; its low sequence diversity is an indicator of an evolutionarily recent population bottleneck. A genome-based phylogenetic reconstruction provides evidence that N. meningitidis has emerged as an unencapsulated human commensal from a common ancestor with Neisseria gonorrhoeae and Neisseria lactamica and consecutively acquired the genes responsible for capsule synthesis via horizontal gene transfer.     

Evolution of host range in Coleosporium ipomoeae,a plant pathogen with multiple hosts

Plants and their pathogens coevolve locally. Previous investigations of one host–one pathogen systems have demonstrated that natural selection favors pathogen genotypes that are virulent on a broad range of host genotypes. In the present study, we examine a system consisting of one pathogen species that infects three host species in the morning glory genus Ipomoea. We show that many pathogen genotypes can infect two or three of the host species when tested on plants from nonlocal communities. By contrast, pathogen genotypes are highly host-specific, infecting only one host species, when tested on host species from the local community. This pattern indicates that within-community evolution narrows the host breadth of pathogen genotypes. Possible evolutionary mechanisms include direct selection for narrow host breadth due to costs of virulence and evolution of ipomoea resistance in the host species.Much of plant-pathogen coevolution is mediated by “gene-for-gene” (GFG) interactions. These interactions involve R genes in plants and corresponding virulence/avirulence genes in the pathogen (1). At a given pair of corresponding loci, a host may carry either a resistant (Res) or a susceptible (Sus) allele, or both, with Res typically being dominant. The pathogen may carry either a virulent (Vir) allele or an avirulent (Avr) allele. Infection results, unless at one pair of corresponding loci, the plant R locus has a Res allele and the pathogen has the Avr allele. Models of the evolution of GFG systems generally predict that generalist pathogens (those able to infect multiple host-resistance genotypes) will be favored by natural selection over highly specialized genotypes that can infect only one resistance genotype (2–6). Experimental analyses of pathogen host breadth in natural plant–pathogen systems are consistent with these expectations in that pathogen isolates are generally able to infect multiple host-resistance genotypes, especially in host populations with high levels of resistance (7–10).With very few exceptions (11, 12), the evolution of pathogen host range has been examined, both theoretically and empirically, for a single pathogen species interacting with a single host species. Many pathogens, however, are capable of infecting multiple host species. Predictions of evolutionary models based on a single evolving host species cannot be clearly extrapolated to this situation. Moreover, there are reasons to believe that, with multiple host species, selection for generalism may not be as prevalent. Maintaining infectivity on multiple hosts requires continued success in the coevolutionary arms race with more than one independently evolving host genome. The conditions under which this maintained infectivity can occur are likely more restrictive than with only one host, although this possibility has not been examined theoretically. In addition, selection to maintain infectivity on a particular host is likely weaker when the pathogen population can successfully reproduce on another host (see ref. 13 for an analogous argument with respect to partial resistance). Finally, costs associated with the ability to infect multiple host species (e.g., ref. 14) are likely greater than costs associated with the ability to infect multiple genotypes within the same host. All of these factors would tend to weaken selection for a broad host range and thus promote the evolution of specialist pathogen genotypes within populations.One approach to determining whether there is an evolutionary tendency for host breadth to be narrowed within populations is to compare pathogen host breadth in its local native community with host breadth on hosts from outside its native community (e.g., refs. 9 and 13). The latter constitutes an estimate of host breadth on host species with which the pathogen has presumably not recently coevolved and is also an estimate of host breadth for a pathogen strain that has recently immigrated into a new community. If evolutionary processes within local communities act to promote specialization, host breadth should be lower on hosts from the native community. In this report, we demonstrate that this pattern is exhibited for a host–pathogen system consisting of one pathogen and three host species.