Journal of Clinical Pharmacy and Therapeutics, 2015, 40, 204–207
doi: 10.1111/jcpt.12245
Comparison of antibiotic susceptibility in viridans group streptococci in low and high antibiotic-prescribing General Practices C. E. Goldsmith* MB BCh BAO BSc, Y. Hara*† BSc MSc, T. Sato*† BSc, T. Nakajima*† BSc MSc PhD, S. Nakanishi*† BSc MSc, C. Mason‡ BSc PhD, J. E. Moore*§ BSc PhD, M. Matsuda† BSc PhD and W. A. Coulter§ BSc MSc BDS PhD *Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast, UK, †Laboratory of Molecular Biology, School of Environmental Health Science, Azabu University, Sagamihara, Japan, ‡School of Dentistry, Queen’s University of Belfast, Royal Group of Hospitals, Belfast, and §School of Biomedical Sciences, University of Ulster, Coleraine, UK
Received 20 August 2014, Accepted 30 December 2014
Keywords: antibiotic resistance, antibiotics, community, fluoroquinolones, infection, macrolide, penicillin, pneumococcus, prescriptions, tetracycline
SUMMARY
WHAT IS KNOWN AND OBJECTIVE
What is known and objective: Antibiotic resistance has become a global public health issue. Most antibiotics are prescribed in the community, although there is less stewardship of such agents in the community compared to secondary and tertiary care. Few studies have attempted to examine the prescribing practices in General Practice and its impact on antibiotic resistance and, therefore, a study was performed in order to compare antibiotic susceptibilities of commensal viridans group streptococci (VGS) obtained from patient cohorts in General Practices (GP), who were high and low prescribers of oral antibiotics. Method: Sixty-five patients (256 044 0064 0064 8 0122 NS
Penicillin G
0047–1 016 0125 0125 05
0094–6 034 025 025 3 0031 *
Ofloxacin
Levofloxacin
075–4 24 2 2 3
038–150 087 1 1 1
025–6 248 3 3 4 0193 NS
050–150 097 1 1 15 0058 NS
* = statistically significant; NS = not significant.
© 2015 John Wiley & Sons Ltd
Journal of Clinical Pharmacy and Therapeutics, 2015, 40, 204–207 205
C. E. Goldsmith et al.
AMR in high vs. low prescribing GP practices
5% [v/v] CO2. Following incubation for 48 h, presumptive VGS isolates (n = 5 per patient2 resembling small and minute blue colonies, as well as ‘gum drop’-like blue colonies and visually distinct morphological variants) were subcultured onto Columbia Blood Agar supplemented with 5% (v/v) defibrinated horse blood for 24 h at 37 °C under microaerophilic conditions, as detailed above. All isolates were subsequently frozen in defibrinated horse blood (2 mL) at 80 °C and stored as part of the MicroARK culture collection (www.microark.com), within the Northern Ireland Public Health Laboratory (NIPHL) Strain Repository.
RESULTS AND DISCUSSION The antibiotic susceptibility of all 65 S. salivarius isolates (29 from low antibiotic-prescribing GP practices + 36 from high antibiotic-prescribing GP practices) was determined by Minimum Inhibitory Concentration (MIC) testing against the five antibiotic agents, as outlined above. The MIC range, geometric MIC, Median and MIC50 and MIC90 values were calculated, summarized and are shown in Table 1. Stepwise unpaired statistical analyses of MIC values obtained from S. salivarius isolates between each patient grouping (low vs. high) was performed and showed that only the penicillin G had a significant signal (P = 0031), whereas there was no association between the other antibiotics and the GP Practices’ prescribing habits. In this study, we employed S. salivarius as the indicator organism of antibiotic resistance. S. salivarius is a common commensal organism of the human mouth and nasopharynx, which is usually non-pathogenic for the human host, although there have been several reports of S. salivarius-related infection.6,7 Phenotypically, this species of streptococci was chosen, as it was the most frequently isolated viridans group streptococcus in the mouth and nasopharynx,8 as well as being easy to isolate, as they produce large mucoid colonies on agar medium containing 5% sucrose, similar to those produced by Leuconostoc species.9,10 The use of several classes of antibiotic agents for the treatment of bacterial respiratory pathogens causing chronic chest infections in patients with cystic fibrosis (CF) has important consequences for the persistence of VGS flora of the treated patient. In order to survive, the VGS organisms colonizing the patient can evolve resistance mechanisms in response to the chronic use of these antibiotic agents. What is not known at present is what resistant mechanisms do commensal organism use and are these mechanisms potentially transferable to hitherto sensitive pathogens? Therefore, antibiotic resistance within the VGS flora of CF patient potentially may be an important reservoir of genetic material for exacerbating antibiotic resistance in CF pathogens. Equally, with the ability to survive intense and prolonged antibiotic pressure, such VGS organisms may become dangerously poised to become potential pathogens, (i) if there is a downward shift in the immune status of the patient, for example following lung transplantation in CF patients; (ii) where such organisms are genetically promiscuous in acquiring virulence determinants from co-habiting with true pathogens; and (iii) where horizontal gene transfer events occur, leading to the acquisition of antibiotic resistance determinants by newly colonizing pathogens. The acquisition of virulence determinants is also a significant cause for concern in antibiotic-resistant VGS organisms and where such commensal flora dominate. One reason for their success is the relative plasticity of their genomes to adapt to varying host immune responses, as well as selective antibiotic pressure. With this genomic plasticity and the ability to naturally transform, VGS organisms have the ability to take up virulence determinants, which then potentially can transform their status from commensal organism to opportunistic pathogen to true pathogen. These study findings are important as the b-lactams are the most commonly prescribed oral antibiotic in the community. High prescribing practices may lead to an altered (higher) level of resistance to these agents in the commensal VGS population, which may be important as reservoirs of antibiotic resistance determinants in subsequent horizontal gene transfer events,
Molecular identification Purified isolates were subcultured on Columbia Blood Agar, as detailed above, for 24 h at 37 °C. All DNA isolation procedures were carried out in a Class II Biological Safety Cabinet (MicroFlow, Andover, Hampshire, UK) in a room physically separated from that used to set up nucleic acid amplification reaction mixes and also from the ‘post-PCR’ room in accordance with the Good Molecular Diagnostic Procedures (GMDP) guidelines of Millar et al.,3 in order to minimize contamination and hence the possibility of false positive results. Bacterial genomic DNA was extracted from few colonies of each purified isolate, by employment of the Roche High Purity PCR Template Preparation Kit (Roche Diagnostics Ltd., Sussex, UK), in accordance with the manufacturer’s instructions. Extracted DNA was stored at 20 °C prior to PCR amplification. Two gene loci were employed to identify the VGS to the species level, namely the rnpB gene4 and the 16S–23S rDNA ITS.4,5 Following PCR amplification, amplicons for sequencing were purified with a QIAquick PCR purification kit (Qiagen Ltd., Manchester, UK) and eluted in TrisHCl (10 mM, pH 85) prior to sequencing. Following labelling of PCR amplicons using Big Dye cycle sequencing chemistry (ABI; Applied Biosystems Ltd., Warrington, UK), automated sequencing was performed on an ABI Capillary Sequencer (3740 platform). Resulting sequences were confirmed from chromatogram analysis, and confirmed sequences were compared with those stored in the GENBANK using the BLASTN alignment software (http://www.blast.genome.ad.jp/). Determination of antimicrobial susceptibility by Minimum Inhibitory Concentration determination Following speciation of all VGS organisms, one isolate belonging to S. salivarius was selected at random, from each individual for subsequent antibiotic susceptibility testing. Antibiotic susceptibility testing was performed on 65 S. salivarius isolates, by standard disc diffusion assay, as well as by e-testing assay (Biomerieux Ltd., Basingstoke, Hampshire, UK), in accordance with the manufacturer’s instructions and against five antibiotics, including the blactams [penicillin G], the tetracyclines [doxycycline], the fluoroquinolones [levofloxacin and ofloxacin] and the macrolides [erythromycin]. Statistical analysis Antibiotic susceptibility was compared statistically within each of the four classes of antibiotic, by comparing the actual zone sizes (mm) of each antibiotic, as determined by disc diffusion methodology, between isolates of S. salivarius obtained from high and low GP practices. An unpaired Student’s t-test was calculated for each population, and a P-value of
doi: 10.1111/jcpt.12245
Comparison of antibiotic susceptibility in viridans group streptococci in low and high antibiotic-prescribing General Practices C. E. Goldsmith* MB BCh BAO BSc, Y. Hara*† BSc MSc, T. Sato*† BSc, T. Nakajima*† BSc MSc PhD, S. Nakanishi*† BSc MSc, C. Mason‡ BSc PhD, J. E. Moore*§ BSc PhD, M. Matsuda† BSc PhD and W. A. Coulter§ BSc MSc BDS PhD *Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast, UK, †Laboratory of Molecular Biology, School of Environmental Health Science, Azabu University, Sagamihara, Japan, ‡School of Dentistry, Queen’s University of Belfast, Royal Group of Hospitals, Belfast, and §School of Biomedical Sciences, University of Ulster, Coleraine, UK
Received 20 August 2014, Accepted 30 December 2014
Keywords: antibiotic resistance, antibiotics, community, fluoroquinolones, infection, macrolide, penicillin, pneumococcus, prescriptions, tetracycline
SUMMARY
WHAT IS KNOWN AND OBJECTIVE
What is known and objective: Antibiotic resistance has become a global public health issue. Most antibiotics are prescribed in the community, although there is less stewardship of such agents in the community compared to secondary and tertiary care. Few studies have attempted to examine the prescribing practices in General Practice and its impact on antibiotic resistance and, therefore, a study was performed in order to compare antibiotic susceptibilities of commensal viridans group streptococci (VGS) obtained from patient cohorts in General Practices (GP), who were high and low prescribers of oral antibiotics. Method: Sixty-five patients (256 044 0064 0064 8 0122 NS
Penicillin G
0047–1 016 0125 0125 05
0094–6 034 025 025 3 0031 *
Ofloxacin
Levofloxacin
075–4 24 2 2 3
038–150 087 1 1 1
025–6 248 3 3 4 0193 NS
050–150 097 1 1 15 0058 NS
* = statistically significant; NS = not significant.
© 2015 John Wiley & Sons Ltd
Journal of Clinical Pharmacy and Therapeutics, 2015, 40, 204–207 205
C. E. Goldsmith et al.
AMR in high vs. low prescribing GP practices
5% [v/v] CO2. Following incubation for 48 h, presumptive VGS isolates (n = 5 per patient2 resembling small and minute blue colonies, as well as ‘gum drop’-like blue colonies and visually distinct morphological variants) were subcultured onto Columbia Blood Agar supplemented with 5% (v/v) defibrinated horse blood for 24 h at 37 °C under microaerophilic conditions, as detailed above. All isolates were subsequently frozen in defibrinated horse blood (2 mL) at 80 °C and stored as part of the MicroARK culture collection (www.microark.com), within the Northern Ireland Public Health Laboratory (NIPHL) Strain Repository.
RESULTS AND DISCUSSION The antibiotic susceptibility of all 65 S. salivarius isolates (29 from low antibiotic-prescribing GP practices + 36 from high antibiotic-prescribing GP practices) was determined by Minimum Inhibitory Concentration (MIC) testing against the five antibiotic agents, as outlined above. The MIC range, geometric MIC, Median and MIC50 and MIC90 values were calculated, summarized and are shown in Table 1. Stepwise unpaired statistical analyses of MIC values obtained from S. salivarius isolates between each patient grouping (low vs. high) was performed and showed that only the penicillin G had a significant signal (P = 0031), whereas there was no association between the other antibiotics and the GP Practices’ prescribing habits. In this study, we employed S. salivarius as the indicator organism of antibiotic resistance. S. salivarius is a common commensal organism of the human mouth and nasopharynx, which is usually non-pathogenic for the human host, although there have been several reports of S. salivarius-related infection.6,7 Phenotypically, this species of streptococci was chosen, as it was the most frequently isolated viridans group streptococcus in the mouth and nasopharynx,8 as well as being easy to isolate, as they produce large mucoid colonies on agar medium containing 5% sucrose, similar to those produced by Leuconostoc species.9,10 The use of several classes of antibiotic agents for the treatment of bacterial respiratory pathogens causing chronic chest infections in patients with cystic fibrosis (CF) has important consequences for the persistence of VGS flora of the treated patient. In order to survive, the VGS organisms colonizing the patient can evolve resistance mechanisms in response to the chronic use of these antibiotic agents. What is not known at present is what resistant mechanisms do commensal organism use and are these mechanisms potentially transferable to hitherto sensitive pathogens? Therefore, antibiotic resistance within the VGS flora of CF patient potentially may be an important reservoir of genetic material for exacerbating antibiotic resistance in CF pathogens. Equally, with the ability to survive intense and prolonged antibiotic pressure, such VGS organisms may become dangerously poised to become potential pathogens, (i) if there is a downward shift in the immune status of the patient, for example following lung transplantation in CF patients; (ii) where such organisms are genetically promiscuous in acquiring virulence determinants from co-habiting with true pathogens; and (iii) where horizontal gene transfer events occur, leading to the acquisition of antibiotic resistance determinants by newly colonizing pathogens. The acquisition of virulence determinants is also a significant cause for concern in antibiotic-resistant VGS organisms and where such commensal flora dominate. One reason for their success is the relative plasticity of their genomes to adapt to varying host immune responses, as well as selective antibiotic pressure. With this genomic plasticity and the ability to naturally transform, VGS organisms have the ability to take up virulence determinants, which then potentially can transform their status from commensal organism to opportunistic pathogen to true pathogen. These study findings are important as the b-lactams are the most commonly prescribed oral antibiotic in the community. High prescribing practices may lead to an altered (higher) level of resistance to these agents in the commensal VGS population, which may be important as reservoirs of antibiotic resistance determinants in subsequent horizontal gene transfer events,
Molecular identification Purified isolates were subcultured on Columbia Blood Agar, as detailed above, for 24 h at 37 °C. All DNA isolation procedures were carried out in a Class II Biological Safety Cabinet (MicroFlow, Andover, Hampshire, UK) in a room physically separated from that used to set up nucleic acid amplification reaction mixes and also from the ‘post-PCR’ room in accordance with the Good Molecular Diagnostic Procedures (GMDP) guidelines of Millar et al.,3 in order to minimize contamination and hence the possibility of false positive results. Bacterial genomic DNA was extracted from few colonies of each purified isolate, by employment of the Roche High Purity PCR Template Preparation Kit (Roche Diagnostics Ltd., Sussex, UK), in accordance with the manufacturer’s instructions. Extracted DNA was stored at 20 °C prior to PCR amplification. Two gene loci were employed to identify the VGS to the species level, namely the rnpB gene4 and the 16S–23S rDNA ITS.4,5 Following PCR amplification, amplicons for sequencing were purified with a QIAquick PCR purification kit (Qiagen Ltd., Manchester, UK) and eluted in TrisHCl (10 mM, pH 85) prior to sequencing. Following labelling of PCR amplicons using Big Dye cycle sequencing chemistry (ABI; Applied Biosystems Ltd., Warrington, UK), automated sequencing was performed on an ABI Capillary Sequencer (3740 platform). Resulting sequences were confirmed from chromatogram analysis, and confirmed sequences were compared with those stored in the GENBANK using the BLASTN alignment software (http://www.blast.genome.ad.jp/). Determination of antimicrobial susceptibility by Minimum Inhibitory Concentration determination Following speciation of all VGS organisms, one isolate belonging to S. salivarius was selected at random, from each individual for subsequent antibiotic susceptibility testing. Antibiotic susceptibility testing was performed on 65 S. salivarius isolates, by standard disc diffusion assay, as well as by e-testing assay (Biomerieux Ltd., Basingstoke, Hampshire, UK), in accordance with the manufacturer’s instructions and against five antibiotics, including the blactams [penicillin G], the tetracyclines [doxycycline], the fluoroquinolones [levofloxacin and ofloxacin] and the macrolides [erythromycin]. Statistical analysis Antibiotic susceptibility was compared statistically within each of the four classes of antibiotic, by comparing the actual zone sizes (mm) of each antibiotic, as determined by disc diffusion methodology, between isolates of S. salivarius obtained from high and low GP practices. An unpaired Student’s t-test was calculated for each population, and a P-value of
