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Papers by Thamarai Schneiders
Journal of Antimicrobial Chemotherapy, 2013
To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF)... more To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF) and non-CF patients and analyse the impact of antibiotic prescribing in the preceding year on resistance amongst CF isolates. The susceptibility of 80 CF Prevotella isolates to 12 antibiotics was compared with that of 50 Prevotella isolates from invasive infections in people who did not have CF and 27 Prevotella isolates from healthy controls. All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam, with only four isolates resistant to metronidazole. However, resistance to amoxicillin, ceftazidime and tetracycline was apparent in all groups. Significant differences in clindamycin resistance (UK CF, 56%; UK invasive, 10%) and co-amoxiclav non-susceptibility (UK CF, 32%; UK invasive, 12%) were observed between UK CF and UK invasive isolates. The likelihood of non-susceptibility to clindamycin and co-amoxiclav in UK CF isolates was 5.5-fold and 2.5-fold higher relative to that in UK invasive isolates, respectively. Azithromycin MICs were also significantly higher for CF isolates (P < 0.001), which was associated with current prescription of azithromycin. More than 50% of clinical isolates tested in this study were β-lactamase positive. This study profiles antibiotic susceptibility in Prevotella spp. in CF and demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated.
Journal of Cystic Fibrosis, 2013
ABSTRACT Objective: The use of chronic azithromcyin treatment has been linked with increased macr... more ABSTRACT Objective: The use of chronic azithromcyin treatment has been linked with increased macrolide resistance. Although, Prevotella spp. are one of the most common anaerobes detected in CF pulmonary samples, the effect of chronic azithromycin exposure on this genus is unknown. The aims of this study were to (i) investigate if resistance is associated with azithromycin prescription (ii) compare azithromcyin and clindamycin MICs between Prevotella isolates cultured from CF patients and healthy control subjects. Methods: Isolates were grouped according to source and patient prescription of azithromycin: CF (currently prescribed), n=27; CF (not currently prescribed), n=30; healthy controls (none), n=17. Susceptibility was determined by Etest® and MICs compared between groups using the Mann-Whitney test. Results: CF isolates had significantly higher azithromycin and clindamycin MICs compared to the healthy control isolates (P<0.001). Current prescription of azithromycin was associated with significantly higher azithromycin MICs (P=0.016) in the CF isolates. CF isolates (not currently prescribed) had significantly higher azithromycin MICs (P=0.009) compared to isolates from healthy control subjects. Isolates from the 2 CF groups had equal resistance to clindamycin (P=0.228). Conclusions: CF patients harbour isolates with increased resistance to azithromycin and clindamycin. Azithromcyin resistance is associated with current azithromycin prescription. Work supported by a Department of Employment and Learning, NI (DEL) studentship to L. Sherrard and by HSC Research and Development, Public Health Agency, NI and the Medical Research Council through a US-Ireland Partnership Grant.
The Journal of antimicrobial chemotherapy, 2014
To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cu... more To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001...
Journal of Cystic Fibrosis, 2012
ABSTRACT Background: Anaerobic conditions exist within the CF lung (1). Potentially pathogenic Pr... more ABSTRACT Background: Anaerobic conditions exist within the CF lung (1). Potentially pathogenic Prevotella spp. have been detected in sputum samples from CF patients in high numbers, from patients at all ages and stages of disease (2). Standard treatment does not target such anaerobes as their role in disease progression is unknown. Aims: (i) To determine in vitro antimicrobial susceptibility of CF and non-CF Prevotella isolates to antibiotics used in the treatment of both CF pulmonary infection and anaerobic infections (ii) to investigate beta-lactamase activity and (iii) identify genes associated with resistance (cfxA/cfxA2, nim and ermF). Methods: Susceptibility of Prevotella isolates from CF (n=41) and non-CF (n=51) patients, to amoxicillin, ceftazidime, clindamycin, co-amoxiclav, meropenem, metronidazole, piperacillin/tazobactam (pip/taz) and tobramycin was determined by E-test®. Beta-lactamase activity was determined using a nitrocefin test. Each isolate was screened for cfxA/cfxA2, nim and ermF genes using PCR assays. Results: Amoxicillin (55%), ceftazidime (30%) and metronidazole (3%) resistance was similar between CF and non-CF isolates. Clindamycin (CF, 56%; non-CF, 10%) and co-amoxiclav (CF, 29%; non-CF, 8 %) resistance was higher among CF isolates. All isolates were sensitive to meropenem and pip/taz but resistant to tobramycin. Beta-lactamase production was detected in 56% (n=41) CF and 52% (n=46) non-CF isolates. Eighty percent (n=56) of isolates were beta-lactamase positive and intermediate/resistant to amoxicillin. Forty-one of 58 (71%) isolates had reduced susceptibility to amoxicillin and were positive for cfxA/cfxA2. Seventeen of 39 (44%) CF isolates were positive for ermF compared to 9/51 (18%) non-CF isolates. Two of 91 (2%) isolates were positive for nim but sensitive to metronidazole. Conclusions: Amoxicillin resistance is common and can be linked with beta-lactamase production and cfxA/cfxA2. Resistance to clindamycin (linked with ermF) and co-amoxiclav was more common among Prevotella isolates from CF patients. Metronidazole, meropenem and pip/taz resistance was not common. Acknowledgements: Supported by a Department of Employment & Learning, NI, studentship to Laura Sherrard. Non-CF Prevotella isolates from the Anaerobe Reference Unit, Public Health Wales, provided by Acting Lead Scientist Dr Val Hall. 1. Worlitzsch D, Tarran R, Ulrich M et al. J. Clin. Invest. 2002;109: 317-325. 2. Tunney MM, Klem ER, Fodor AA et al. Thorax. 2011; 66: 579-584.
Journal of Cystic Fibrosis, 2013
International Journal of Antimicrobial Agents, 2011
Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulatio... more Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA-ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA-ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA-ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA-ramA genes, strongly suggesting that a secondary regulator may control ramA expression.
Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported previo... more Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported previously from Pakistan but the genotypic characteristics of these enzymes is not known. Hence the aim of the study was first to characterise the genotypic content of these beta-lactamases and secondly to assess the clonal relationship of these isolates. We analysed 65 non-duplicate ESBL positive, K. pneumoniae isolates prospectively collected based on phenotype as detected using the two-disc method. Isolates were collected from different sources: blood cultures (46.15%; n = 30); tracheal aspirates (24.6%; n = 16); urine (10.7%; n = 7); wound swabs, pus and tissue (18.4%; n = 12). ESBL production was confirmed by the ESBL E-test method and the presence of the blaCTX-M encoding genes was confirmed by polymerase chain reaction. The clonal relationship of clinical isolates was studied by Pulsed Field Gel Electrophoresis. The results showed that 93.84% (n = 61) isolates of K. pneumoniae were positive for the blaCTX-M-1 group. One isolate showed PCR signals for blaCTX-M-25 group. None of our isolates were positive for CTX-M groups 2, 8 and 9. The majority of blaCTX-M positive isolates were genetically unrelated and no epidemic clones were identified. This study reports the emergence of CTX-M groups 1 and 25 producing isolates of K. pneumoniae with genetic diversity in Karachi, Pakistan.
PLOS Pathogens, 2015
Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug res... more Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.
Journal of Antimicrobial Chemotherapy, 2013
To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF)... more To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF) and non-CF patients and analyse the impact of antibiotic prescribing in the preceding year on resistance amongst CF isolates. The susceptibility of 80 CF Prevotella isolates to 12 antibiotics was compared with that of 50 Prevotella isolates from invasive infections in people who did not have CF and 27 Prevotella isolates from healthy controls. All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam, with only four isolates resistant to metronidazole. However, resistance to amoxicillin, ceftazidime and tetracycline was apparent in all groups. Significant differences in clindamycin resistance (UK CF, 56%; UK invasive, 10%) and co-amoxiclav non-susceptibility (UK CF, 32%; UK invasive, 12%) were observed between UK CF and UK invasive isolates. The likelihood of non-susceptibility to clindamycin and co-amoxiclav in UK CF isolates was 5.5-fold and 2.5-fold higher relative to that in UK invasive isolates, respectively. Azithromycin MICs were also significantly higher for CF isolates (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001), which was associated with current prescription of azithromycin. More than 50% of clinical isolates tested in this study were β-lactamase positive. This study profiles antibiotic susceptibility in Prevotella spp. in CF and demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated.
Journal of Cystic Fibrosis, 2013
ABSTRACT Objective: The use of chronic azithromcyin treatment has been linked with increased macr... more ABSTRACT Objective: The use of chronic azithromcyin treatment has been linked with increased macrolide resistance. Although, Prevotella spp. are one of the most common anaerobes detected in CF pulmonary samples, the effect of chronic azithromycin exposure on this genus is unknown. The aims of this study were to (i) investigate if resistance is associated with azithromycin prescription (ii) compare azithromcyin and clindamycin MICs between Prevotella isolates cultured from CF patients and healthy control subjects. Methods: Isolates were grouped according to source and patient prescription of azithromycin: CF (currently prescribed), n=27; CF (not currently prescribed), n=30; healthy controls (none), n=17. Susceptibility was determined by Etest® and MICs compared between groups using the Mann-Whitney test. Results: CF isolates had significantly higher azithromycin and clindamycin MICs compared to the healthy control isolates (P<0.001). Current prescription of azithromycin was associated with significantly higher azithromycin MICs (P=0.016) in the CF isolates. CF isolates (not currently prescribed) had significantly higher azithromycin MICs (P=0.009) compared to isolates from healthy control subjects. Isolates from the 2 CF groups had equal resistance to clindamycin (P=0.228). Conclusions: CF patients harbour isolates with increased resistance to azithromycin and clindamycin. Azithromcyin resistance is associated with current azithromycin prescription. Work supported by a Department of Employment and Learning, NI (DEL) studentship to L. Sherrard and by HSC Research and Development, Public Health Agency, NI and the Medical Research Council through a US-Ireland Partnership Grant.
The Journal of antimicrobial chemotherapy, 2014
To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cu... more To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001...
Journal of Cystic Fibrosis, 2012
ABSTRACT Background: Anaerobic conditions exist within the CF lung (1). Potentially pathogenic Pr... more ABSTRACT Background: Anaerobic conditions exist within the CF lung (1). Potentially pathogenic Prevotella spp. have been detected in sputum samples from CF patients in high numbers, from patients at all ages and stages of disease (2). Standard treatment does not target such anaerobes as their role in disease progression is unknown. Aims: (i) To determine in vitro antimicrobial susceptibility of CF and non-CF Prevotella isolates to antibiotics used in the treatment of both CF pulmonary infection and anaerobic infections (ii) to investigate beta-lactamase activity and (iii) identify genes associated with resistance (cfxA/cfxA2, nim and ermF). Methods: Susceptibility of Prevotella isolates from CF (n=41) and non-CF (n=51) patients, to amoxicillin, ceftazidime, clindamycin, co-amoxiclav, meropenem, metronidazole, piperacillin/tazobactam (pip/taz) and tobramycin was determined by E-test®. Beta-lactamase activity was determined using a nitrocefin test. Each isolate was screened for cfxA/cfxA2, nim and ermF genes using PCR assays. Results: Amoxicillin (55%), ceftazidime (30%) and metronidazole (3%) resistance was similar between CF and non-CF isolates. Clindamycin (CF, 56%; non-CF, 10%) and co-amoxiclav (CF, 29%; non-CF, 8 %) resistance was higher among CF isolates. All isolates were sensitive to meropenem and pip/taz but resistant to tobramycin. Beta-lactamase production was detected in 56% (n=41) CF and 52% (n=46) non-CF isolates. Eighty percent (n=56) of isolates were beta-lactamase positive and intermediate/resistant to amoxicillin. Forty-one of 58 (71%) isolates had reduced susceptibility to amoxicillin and were positive for cfxA/cfxA2. Seventeen of 39 (44%) CF isolates were positive for ermF compared to 9/51 (18%) non-CF isolates. Two of 91 (2%) isolates were positive for nim but sensitive to metronidazole. Conclusions: Amoxicillin resistance is common and can be linked with beta-lactamase production and cfxA/cfxA2. Resistance to clindamycin (linked with ermF) and co-amoxiclav was more common among Prevotella isolates from CF patients. Metronidazole, meropenem and pip/taz resistance was not common. Acknowledgements: Supported by a Department of Employment & Learning, NI, studentship to Laura Sherrard. Non-CF Prevotella isolates from the Anaerobe Reference Unit, Public Health Wales, provided by Acting Lead Scientist Dr Val Hall. 1. Worlitzsch D, Tarran R, Ulrich M et al. J. Clin. Invest. 2002;109: 317-325. 2. Tunney MM, Klem ER, Fodor AA et al. Thorax. 2011; 66: 579-584.
Journal of Cystic Fibrosis, 2013
International Journal of Antimicrobial Agents, 2011
Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulatio... more Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA-ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA-ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA-ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA-ramA genes, strongly suggesting that a secondary regulator may control ramA expression.
Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported previo... more Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported previously from Pakistan but the genotypic characteristics of these enzymes is not known. Hence the aim of the study was first to characterise the genotypic content of these beta-lactamases and secondly to assess the clonal relationship of these isolates. We analysed 65 non-duplicate ESBL positive, K. pneumoniae isolates prospectively collected based on phenotype as detected using the two-disc method. Isolates were collected from different sources: blood cultures (46.15%; n = 30); tracheal aspirates (24.6%; n = 16); urine (10.7%; n = 7); wound swabs, pus and tissue (18.4%; n = 12). ESBL production was confirmed by the ESBL E-test method and the presence of the blaCTX-M encoding genes was confirmed by polymerase chain reaction. The clonal relationship of clinical isolates was studied by Pulsed Field Gel Electrophoresis. The results showed that 93.84% (n = 61) isolates of K. pneumoniae were positive for the blaCTX-M-1 group. One isolate showed PCR signals for blaCTX-M-25 group. None of our isolates were positive for CTX-M groups 2, 8 and 9. The majority of blaCTX-M positive isolates were genetically unrelated and no epidemic clones were identified. This study reports the emergence of CTX-M groups 1 and 25 producing isolates of K. pneumoniae with genetic diversity in Karachi, Pakistan.
PLOS Pathogens, 2015
Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug res... more Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.