Phenotypic and genotypic characterisation of Neisseria gohorrhoeae isolates from New Zealand with reduced susceptibility to ceftriaxone : a thesis submitted to the College of Health in partial fulfilment of the requirements for the Master of Science in Microbiology at Massey University, New Zealand

Objectives Currently, ceftriaxone is the last remaining drug recommended for empirical treatment of gonorrhoea. Neisseria gonorrhoeae with reduced susceptibility to ceftriaxone have been isolated worldwide in countries such as Japan, France, Spain, Slovenia, Australia and Sweden. These have led to treatment failures and the emergence of ceftriaxone-resistant N. gonorrhoeae. Various mutations in penA (mosaic and nonmosaic), which encodes the penicillin-binding protein 2 (PBP2), have been reported to be the primary reason for reduced ceftriaxone susceptibility, but it can be reduced further by mutations in mtrR, porBIB and ponA. In this study, we aimed to determine the antimicrobial resistance patterns of New Zealand isolates of N. gonorrhoeae with reduced susceptibility to ceftriaxone and to characterise the penA, mtrR, porBIB and ponA in the isolates. Methods A total of 28 N. gonorrhoeae isolates with elevated ceftriaxone MIC (0.03 to 0.12 mg/L), collected from 2012 to 2015 and obtained from the Institute of Environmental Science and Research (ESR), were examined in this study. Samples came from laboratories in Auckland (26), Wellington (1) and Taranaki (1). The antimicrobial resistance of penicillin G, tetracycline, ciprofloxacin, azithromycin and ceftriaxone were determined through antimicrobial susceptibility test, using minimum inhibitory concentration (MIC) test strips. Polymerase chain reactions (PCRs) and sequencing to identify specific mutations in penA, mtrR, porBIB and ponA, that are associated with elevated minimum inhibitory concentrations (MICs) to ceftriaxone, were undertaken. The association between the phenotypic and genotypic results was investigated by comparing the presence of the number of mutated genes and the MIC level of ceftriaxone. Results Based on the AST results using MIC test strips and interpreted using The European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria, 23 out of 28 isolates (82%) showed reduced susceptibility to ceftriaxone, with MICs of 0.03 to 0.06 mg/L. All of the isolates were resistant to ciprofloxacin, while 36%, 25% and 7% were resistant to penicillin G, tetracycline and azithromycin, respectively. Two azithromycin-resistant N. gonorrhoeae isolates were observed, and isolate 264 (azithromycin MIC: 4mg/L) also exhibited reduced susceptibility to ceftriaxone (MIC: 0.03 mg/L). A total of 21% (6/28) of the isolates produced ß- lactamase. The 23 isolates that conveyed reduced ceftriaxone susceptibility were found to harbour three or four mutated genes (penA, mtrR and/or porBIB and ponA). Reduced susceptibility to ceftriaxone among N. gonorrhoeae isolates in this study was associated with mosaic PBP2 (encoded by penA) with G545S/A501V mutations, with nonmosaic PBP2 with an A501V mutation, plus the presence of mutation in mtrR promoter with G120 and A121 alterations in PorBIB. A total of 65% (15/23) of the N. gonorrhoeae isolates with reduced susceptibility to ceftriaxone harboured mosaic PBP2 XXXIV, a pattern found in N. gonorrhoeae associated with ceftriaxone treatment failures in Europe and Australia. The current study also revealed that the partial sequences of four mosaic PBP2 (M-2, M-3, M-4, M-5) were different from the common mosaic PBP2 sequences reported in various studies. Conclusion There is an association between the phenotypic and genotypic character of N. gonorrhoeae isolates expressing reduced susceptibility to ceftriaxone in this study population. Furthermore, the presence of important mosaic PBP2 that link to ceftriaxone treatment failure might be circulating among N. gonorrhoeae isolates in New Zealand . Keywords: Neisseria gonorrhoeae, ceftriaxone, reduced susceptibility, New Zealan

Characterization of shifts of koala (Phascolarctos cinereus) intestinal microbial communities associated with antibiotic treatment.

Koalas (Phascolarctos cinereus) are arboreal marsupials native to Australia that eat a specialized diet of almost exclusively eucalyptus leaves. Microbes in koala intestines are known to break down otherwise toxic compounds, such as tannins, in eucalyptus leaves. Infections by Chlamydia, obligate intracellular bacterial pathogens, are highly prevalent in koala populations. If animals with Chlamydia infections are received by wildlife hospitals, a range of antibiotics can be used to treat them. However, previous studies suggested that koalas can suffer adverse side effects during antibiotic treatment. This study aimed to use 16S rRNA gene sequences derived from koala feces to characterize the intestinal microbiome of koalas throughout antibiotic treatment and identify specific taxa associated with koala health after treatment. Although differences in the alpha diversity were observed in the intestinal flora between treated and untreated koalas and between koalas treated with different antibiotics, these differences were not statistically significant. The alpha diversity of microbial communities from koalas that lived through antibiotic treatment versus those who did not was significantly greater, however. Beta diversity analysis largely confirmed the latter observation, revealing that the overall communities were different between koalas on antibiotics that died versus those that survived or never received antibiotics. Using both machine learning and OTU (operational taxonomic unit) co-occurrence network analyses, we found that OTUs that are very closely related to Lonepinella koalarum, a known tannin degrader found by culture-based methods to be present in koala intestines, was correlated with a koala's health status. This is the first study to characterize the time course of effects of antibiotics on koala intestinal microbiomes. Our results suggest it may be useful to pursue alternative treatments for Chlamydia infections without the use of antibiotics or the development of Chlamydia-specific antimicrobial compounds that do not broadly affect microbial communities

Methylation Status of Imprinted Genes and Repetitive Elements in Sperm DNA from Infertile Males

Stochastic, environmentally and/or genetically induced disturbances in the genome-wide epigenetic reprogramming processes during male germ-cell development may contribute to male infertility. To test this hypothesis, we have studied the methylation levels of 2 paternally (H19 and GTL2) and 5 maternally methylated (LIT1, MEST, NESPAS, PEG3, and SNRPN) imprinted genes, as well as of ALU and LINE1 repetitive elements in 141 sperm samples, which were used for assisted reproductive technologies (ART), including 106 couples with strictly male-factor or combined male and female infertility and 28 couples with strictly female-factor infertility. Aberrant methylation imprints showed a significant association with abnormal semen parameters, but did not seem to influence ART outcome. Repeat methylation also differed significantly between sperm samples from infertile and presumably fertile males. However, in contrast to imprinted genes, ALU methylation had a significant impact on pregnancy and live-birth rate in couples with male-factor or combined infertility. ALU methylation was significantly high-er in sperm samples leading to pregnancy and live-birth than in those that did not. Sperm samples leading to abortions showed significantly lower ALU methylation levels than those leading to the birth of a baby. Copyright (C) 2011 S. Karger AG, Base

MicroRNA-like RNAs from the same miRNA precursors play a role in cassava chilling responses

Abstract MicroRNAs (miRNAs) are known to play important roles in various cellular processes and stress responses. MiRNAs can be identified by analyzing reads from high-throughput deep sequencing. The reads realigned to miRNA precursors besides canonical miRNAs were initially considered as sequencing noise and ignored from further analysis. Here we reported a small-RNA species of phased and half-phased miRNA-like RNAs different from canonical miRNAs from cassava miRNA precursors detected under four distinct chilling conditions. They can form abundant multiple small RNAs arranged along precursors in a tandem and phased or half-phased fashion. Some of these miRNA-like RNAs were experimentally confirmed by re-amplification and re-sequencing, and have a similar qRT-PCR detection ratio as their cognate canonical miRNAs. The target genes of those phased and half-phased miRNA-like RNAs function in process of cell growth metabolism and play roles in protein kinase. Half-phased miR171d.3 was confirmed to have cleavage activities on its target gene P-glycoprotein 11, a broad substrate efflux pump across cellular membranes, which is thought to provide protection for tropical cassava during sharp temperature decease. Our results showed that the RNAs from miRNA precursors are miRNA-like small RNAs that are viable negative gene regulators and may have potential functions in cassava chilling responses

European mtDNA Variants Are Associated With Differential Responses to Cisplatin, an Anticancer Drug: Implications for Drug Resistance and Side Effects.

Background: Cisplatin, a powerful antitumor agent, causes formation of DNA adducts, and activation of apoptotic pathways. Presently, cisplatin resistance develops in up to 70% of patients but the underlying molecular mechanism(s) are unclear and there are no markers to determine which patients will become resistant. Mitochondria play a significant role not only in energy metabolism but also retrograde signaling (mitochondria to nucleus) that modulates inflammation, complement, and apoptosis pathways. Maternally inherited mitochondrial (mt) DNA can be classified into haplogroups representing different ethnic populations that have diverse susceptibilities to diseases and medications. Methods: Transmitochondrial cybrids, where all cell lines possess identical nuclear genomes but either the H (Southern European) or J (Northern European) mtDNA haplogroups, were treated with cisplatin and analyzed for differential responses related to viability, oxidative stress, and expression levels of genes associated with cancer, cisplatin-induced nephrotoxicity and resistance, apoptosis and signaling pathways. Results: The cisplatin-treated-J cybrids showed greater loss of cell viability along with lower levels of reactive oxygen species and mitochondrial membrane potential compared to cisplatin-treated-H cybrids. After cisplatin treatment, J cybrids showed increased gene expression of BAX, CASP3, and CYP51A, but lower levels of SFRP1 compared to untreated-J cybrids. The cisplatin-treated-H cybrids had elevated expression of CDKN1A/P21, which has a role in cisplatin toxicity, compared to untreated-H cybrids. The cisplatin-treated H had higher transcription levels of ABCC1, DHRS2/HEP27, and EFEMP1 compared to cisplatin-treated-J cybrids. Conclusions: Cybrid cell lines that contain identical nuclei but either H mtDNA mitochondria or J mtDNA mitochondria respond differently to cisplatin treatments suggesting involvement of the retrograde signaling (from mitochondria to nucleus) in the drug-induced cell death. Varying toxicities and transcription levels of the H vs. J cybrids after cisplatin treatment support the hypothesis that mtDNA variants play a role in the expression of genes affecting resistance and side effects of cisplatin

Quantitative Comparison of Abundance Structures of Generalized Communities: From B-Cell Receptor Repertoires to Microbiomes

The \emph{community}, the assemblage of organisms co-existing in a given space and time, has the potential to become one of the unifying concepts of biology, especially with the advent of high-throughput sequencing experiments that reveal genetic diversity exhaustively. In this spirit we show that a tool from community ecology, the Rank Abundance Distribution (RAD), can be turned by the new MaxRank normalization method into a generic, expressive descriptor for quantitative comparison of communities in many areas of biology. To illustrate the versatility of the method, we analyze RADs from various \emph{generalized communities}, i.e.\ assemblages of genetically diverse cells or organisms, including human B cells, gut microbiomes under antibiotic treatment and of different ages and countries of origin, and other human and environmental microbial communities. We show that normalized RADs enable novel quantitative approaches that help to understand structures and dynamics of complex generalize communities

Comparison of TCGA and GENIE genomic datasets for the detection of clinically actionable alterations in breast cancer.

Whole exome sequencing (WES), targeted gene panel sequencing and single nucleotide polymorphism (SNP) arrays are increasingly used for the identification of actionable alterations that are critical to cancer care. Here, we compared The Cancer Genome Atlas (TCGA) and the Genomics Evidence Neoplasia Information Exchange (GENIE) breast cancer genomic datasets (array and next generation sequencing (NGS) data) in detecting genomic alterations in clinically relevant genes. We performed an in silico analysis to determine the concordance in the frequencies of actionable mutations and copy number alterations/aberrations (CNAs) in the two most common breast cancer histologies, invasive lobular and invasive ductal carcinoma. We found that targeted sequencing identified a larger number of mutational hotspots and clinically significant amplifications that would have been missed by WES and SNP arrays in many actionable genes such as PIK3CA, EGFR, AKT3, FGFR1, ERBB2, ERBB3 and ESR1. The striking differences between the number of mutational hotspots and CNAs generated from these platforms highlight a number of factors that should be considered in the interpretation of array and NGS-based genomic data for precision medicine. Targeted panel sequencing was preferable to WES to define the full spectrum of somatic mutations present in a tumor

Epigenetics as a mechanism driving polygenic clinical drug resistance

Aberrant methylation of CpG islands located at or near gene promoters is associated with inactivation of gene expression during tumour development. It is increasingly recognised that such epimutations may occur at a much higher frequency than gene mutation and therefore have a greater impact on selection of subpopulations of cells during tumour progression or acquisition of resistance to anticancer drugs. Although laboratory-based models of acquired resistance to anticancer agents tend to focus on specific genes or biochemical pathways, such 'one gene : one outcome' models may be an oversimplification of acquired resistance to treatment of cancer patients. Instead, clinical drug resistance may be due to changes in expression of a large number of genes that have a cumulative impact on chemosensitivity. Aberrant CpG island methylation of multiple genes occurring in a nonrandom manner during tumour development and during the acquisition of drug resistance provides a mechanism whereby expression of multiple genes could be affected simultaneously resulting in polygenic clinical drug resistance. If simultaneous epigenetic regulation of multiple genes is indeed a major driving force behind acquired resistance of patients' tumour to anticancer agents, this has important implications for biomarker studies of clinical outcome following chemotherapy and for clinical approaches designed to circumvent or modulate drug resistance