Project description:Streptococcus suis is a pathogen that causes invasive infections in humans and pigs. In this study, 448 S. suis isolates recovered from human infections in Thailand were characterized with regard to their antimicrobial susceptibility and antimicrobial resistance genes, including, for non-penicillin-susceptible isolates, sequence analyses of five genes encoding penicillin-binding proteins (pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x). All 448 isolates were susceptible to cefepime and ceftriaxone, whereas 99.6%, 91.7%, and 72.9% of the isolates were susceptible to levofloxacin, penicillin, and chloramphenicol, respectively. Almost all isolates were resistant to tetracycline (98.2%), clindamycin (94%), erythromycin (92.4%), and azithromycin (82.6%). Genes tet(O) and ermB were the predominant resistance genes detected among macrolide- and tetracycline-resistant isolates. A total of 37 out of 448 isolates (8.2%) showed intermediately resistance to penicillin. Most of these isolates (59.5%) belonged to serotype 2-ST233. Comparison of the predicted translated sequences of five PBP proteins of a penicillin-susceptible isolate (strain P1/7) to the respective PBP sequences of ten non-penicillin-susceptible isolates revealed multiple amino acid substitutions. Isolates of CC221/234 showed highly variable amino acid substitutions in all PBP proteins. An ST104 isolate had a higher number of amino acid substitutions in PBP2X. Isolates belonging to CC233/379 had numerous substitutions in PBP2B and PBP2X. ST25 isolates exhibited fewer amino acid substitutions than isolates of other STs in all five PBPs. The antimicrobial resistance of S. suis is increasing worldwide; therefore, restrictions on antimicrobial use, continuous control, and the surveillance of this bacterium throughout the pork supply chain are crucial for ensuring public health and must be a priority concern.
Project description:The increasing rate of penicillin resistance in S. pneumoniae in the early 1970s has resulted in therapeutic challenges and has prompted the need for alternative therapy in the management of pneumococcal infections. The development of penicillin resistance has been documented to be as a result of altered penicillin binding protein which alters the binding capacity of the drug to the organism. We used microarrays to investigate other genes which may be involved in the development of penicillin resistance in S. pneumoniae and identified classes of genes on the surface of the organism which may contribute to resistance.
Project description:The increasing rate of penicillin resistance in S. pneumoniae in the early 1970s has resulted in therapeutic challenges and has prompted the need for alternative therapy in the management of pneumococcal infections. The development of penicillin resistance has been documented to be as a result of altered penicillin binding protein which alters the binding capacity of the drug to the organism. We used microarrays to investigate other genes which may be involved in the development of penicillin resistance in S. pneumoniae and identified classes of genes on the surface of the organism which may contribute to resistance. Strains of S. pneumoniae with varying initial susceptibility to penicillin were selected. These strains were grown to the logarithmic phase before being exposed to subinhibitory concentration of penicillin. RNA was extracted before and after penicillin stress and hybridized on Affymetrix microarrays and represented as either Untreated (before penicillin stress) or treated (after penicillin stress). This was carried out for 3 representative strains; S676, I81, and R98. S, I, and R abbreviates Sensitive, Intermediate and resistant to Penicillin.
Project description:ObjectivesStreptococcus agalactiae [group B streptococci (GBS)] have been considered uniformly susceptible to penicillin. However, increasing reports from Asia and North America are documenting penicillin-non-susceptible GBS (PRGBS) with mutations in pbp genes. Here we report, to the best of our knowledge, the first two PRGBS isolates recovered in Europe (AC-13238-1 and AC-13238-2), isolated from the same patient.MethodsTwo different colony morphologies of GBS were noted from a surgical abscess drainage sample. Both were serotyped and antimicrobial susceptibility testing was performed by different methodologies. High-throughput sequencing was done to compare the isolates at the genomic level, to identify their capsular type and ST, to evaluate mutations in the pbp genes and to compare the isolates with the genomes of other PRGBS isolates sharing the same serotype and ST.ResultsIsolates AC-13238-1 and AC-13238-2 presented MICs above the EUCAST and CLSI breakpoints for penicillin susceptibility. Both shared the capsular type Ia operon and ST23. Genomic analysis uncovered differences between the two isolates in seven genes, including altered pbp genes. Deduced amino acid sequences revealed critical substitutions in PBP2X in both isolates. Comparison with serotype Ia clonal complex 23 PRGBS from the USA reinforced the similarity between AC-13238-1 and AC-13238-2, and their divergence from the US strains.ConclusionsOur results support the in-host evolution of β-lactam-resistant GBS, with two PRGBS variants being isolated from one patient.
Project description:Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease microbes. To provide a new tool to study AMR, here we develop a Klebsiella pneumoniae cell-free gene expression (CFE) system. To characterise the system, we use proteomics to compare this to a Escherichia coli MG1655 CFE model, to identify relative differences and unique proteins. Then we use this native CFE system to profile antimicrobial activity in comparison to whole cell inhibition, to reveal host differences in IC50/MIC50 values. Finally, we use the CFE tool to study AMR variants, at a proof-of-concept level. As an exemplar, we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin – a common genotype frequently observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. In summary, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial processing requires Biosafety Level 2, the final extracts are non-living and suitable for long-term storage, and potentially transfer to a Biosafety Level 1 lab. This bioassay has potential uses for early-stage host-specific antimicrobial development and the testing of AMR variants for structure-activity relationship studies. The data reposited is label-free high-resolution LC-MS proteomics data performed to characterise the proteins in cell-free extract of K. pneumoniae ATCC 13882 and compare to that of E. coli MG1655 to identify common and unique proteins. We also characterised the proteins of K. pneumoniae clinically resistant isolates ST258-T1b and NJST258-1, and compared them to K. pneumoniae ATCC 13882 laboratory strain.
Project description:Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease microbes. To provide a new tool to study AMR, here we develop a Klebsiella pneumoniae cell-free gene expression (CFE) system. To characterise the system, we use proteomics to compare this to a Escherichia coli MG1655 CFE model, to identify relative differences and unique proteins. Then we use this native CFE system to profile antimicrobial activity in comparison to whole cell inhibition, to reveal host differences in IC50/MIC50 values. Finally, we use the CFE tool to study AMR variants, at a proof-of-concept level. As an exemplar, we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin – a common genotype frequently observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. In summary, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial processing requires Biosafety Level 2, the final extracts are non-living and suitable for long-term storage, and potentially transfer to a Biosafety Level 1 lab. This bioassay has potential uses for early-stage host-specific antimicrobial development and the testing of AMR variants for structure-activity relationship studies. The data reposited is label-free high-resolution LC-MS proteomics data performed to characterise the proteins in cell-free extract of K. pneumoniae ATCC 13882 and compare to that of E. coli MG1655 to identify common and unique proteins. We also characterised the proteins of K. pneumoniae clinically resistant isolates ST258-T1b and NJST258-1, and compared them to K. pneumoniae ATCC 13882 laboratory strain.
Project description:BackgroundOver one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing.MethodsWe assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP.ResultsFifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLSB phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic correlates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes.ConclusionThe erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLSB in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon.