Transient Silencing of Antibiotic Resistance by Mutation Represents a Significant Potential Source of Unanticipated Therapeutic Failure.
ABSTRACT: Sporadic literature reports describe isolates of pathogenic bacteria that harbor an antibiotic resistance determinant but remain susceptible to the corresponding antibiotic as a consequence of a genetic defect. Such strains represent a source from which antibiotic resistance may reemerge to cause treatment failure in patients. Here, we report a systematic investigation into the prevalence and nature of this phenomenon, which we term silencing of antibiotic resistance by mutation (SARM). Instances of SARM were detected among 1,470 Staphylococcus aureus isolates through side-by-side comparison of antibiotic resistance genotype (as determined by whole-genome sequencing) versus phenotype (as assessed through susceptibility testing). Of the isolates analyzed, 152 (10.3%) harbored a silenced resistance gene, including 46 (3.1%) that exhibited SARM to currently deployed antistaphylococcal drugs. SARM resulted from diverse mutational events but most commonly through frameshift mutation of resistance determinants as a result of point deletion in poly(A) tracts. The majority (?90%) of SARM strains reverted to antibiotic resistance at frequencies of ?10-9; thus, while appearing antibiotic sensitive in the clinical microbiology laboratory, most S. aureus isolates exhibiting SARM will revert to antibiotic resistance at frequencies achievable in patients. In view of its prevalence in a major pathogen, SARM represents a significant potential threat to the therapeutic efficacy of antibiotics.IMPORTANCE Antibiotic resistance hinders the treatment of bacterial infection. To guide effective therapy, clinical microbiology laboratories routinely perform susceptibility testing to determine the antibiotic sensitivity of an infecting pathogen. This approach relies on the assumption that it can reliably distinguish bacteria capable of expressing antibiotic resistance in patients, an idea challenged by the present study. We report that the important human pathogen Staphylococcus aureus frequently carries antibiotic resistance genes that have become inactivated ("silenced") by mutation, leading strains to appear antibiotic sensitive. However, resistance can rapidly reemerge in most such cases, at frequencies readily achievable in infected patients. Silent antibiotic resistance is therefore prevalent, transient, and evades routine detection, rendering it a significant potential threat to antibacterial chemotherapy.
Project description:The Antibiotic Resistance Monitoring in Ocular Microorganisms (ARMOR) surveillance study evaluates in vitro antibiotic resistance among Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Streptococcus pneumoniae, Pseudomonas aeruginosa, and Haemophilus influenzae isolates from ocular infections. Here we report resistance rates and trends among conjunctival-sourced ocular isolates collected across the US from 2009 through 2016. A total of 1198 conjunctival isolates (483 S. aureus, 305 CoNS, 208 H. influenzae, 118 S. pneumoniae, and 84 P. aeruginosa) were collected from patients with presumed bacterial conjunctivitis from 57 sites across 40 states. A large proportion of staphylococci demonstrated resistance to oxacillin and azithromycin, while resistance was low against the majority of antibiotics tested for S. pneumoniae, P. aeruginosa, and H. influenzae. Multidrug resistance (?3 antibiotic classes) was found in 30.2% of S. aureus and 39.0% of CoNS isolates, and methicillin resistance more than doubled the rate of multi-drug resistance (methicillin-resistant S. aureus [MRSA], 76.5%; methicillin-resistant CoNS isolates, 72.8%). There was a pattern of increasing mean percent resistance with increasing age by decade of life among S. aureus, MRSA, and CoNS (P?0.038). Over the eight-year study period, there were small yet significant decreases in resistance rates among S. aureus to azithromycin, ciprofloxacin, tobramycin, trimethoprim, and oxacillin (P?0.003), and among CoNS and P. aeruginosa (both P<0.05) to ciprofloxacin. These data indicate that antibiotic resistance is high, but did not increase, among conjunctival-sourced isolates collected in the US from 2009 through 2016. For certain antibiotic/pathogen combinations, there was a trend of decreased resistance, including a decrease in oxacillin resistance among S. aureus.
Project description:AIMS:To report trends in antibiotic resistance in cases of bacterial keratitis from a large eye hospital in South India. METHODS:In this retrospective cross-sectional study, the microbiology laboratory records of patients with infectious keratitis diagnosed at an eye hospital in South India from 2002 to 2013 were reviewed to determine the proportion with antibiotic non-susceptibility. RESULTS:3685 bacterial isolates had susceptibility testing performed over the 12-year period. The two most common organisms with resistance were Streptococcus pneumoniae (n=1204) and Pseudomonas aeruginosa (n=894). Antibiotic non-susceptibility was generally uncommon for these two organisms and no significant trends were detected over the course of the study. In contrast, Staphylococcus aureus (N=211) isolates demonstrated a significant increase in fluoroquinolone non-susceptibility over the 12-year study period. This coincided with a significant increase in methicillin-resistant S. aureus (MRSA) during the study period, though the increase in fluoroquinolone resistance was likewise seen in methicillin-sensitive S. aureus (MSSA). For example, ofloxacin resistance in MSSA increased from 11.1% in 2002 to 66.7% in 2013 (p=0.002). No trends were apparent for the aminoglycosides, cefazolin or vancomycin, for which in vitro non-susceptibility generally appeared to be low. CONCLUSION:Resistance to antibiotics was generally stable for infectious keratitis isolates from a large eye hospital in South India, except for S. aureus, which experienced a significant increase in fluoroquinolone resistance from 2002 to 2013. Fluoroquinolone antibiotics currently have poor in vitro activity against both MRSA and MSSA in South India and are therefore not the ideal therapy for Staphylococcal corneal ulcers.
Project description:Rapid bacterial antibiotic susceptibility test (AST) and minimum inhibitory concentration (MIC) measurement are important to help reduce the widespread misuse of antibiotics and alleviate the growing drug-resistance problem. We discovered that, when a susceptible strain of Staphylococcus aureus or Escherichia coli is exposed to an antibiotic, the intensity of specific biomarkers in its surface-enhanced Raman scattering (SERS) spectra drops evidently in two hours. The discovery has been exploited for rapid AST and MIC determination of methicillin-susceptible S. aureus and wild-type E. coli as well as clinical isolates. The results obtained by this SERS-AST method were consistent with that by the standard incubation-based method, indicating its high potential to supplement or replace existing time-consuming methods and help mitigate the challenge of drug resistance in clinical microbiology.
Project description:Aim:The extent of methicillin-resistant Staphylococcus aureus (MRSA) infection in Nepalese children is largely unknown. Materials & methods:Six hundred and seventy-two clinical samples collected from 232 patients between June and November 2016 were processed in a microbiology laboratory. Results:Out of 300 culture-positive samples, 52 (17.3%) were S. aureus isolates. Among those 52, 39 (75.0%) were found to be MRSA. The infection rate of S. aureus was shown to be higher in inpatients (55.7%) compared with outpatients (44.3%) at p = 0.637, 95% CI. Thirteen types of antibiotics were used in the antibiotic susceptibility test. MRSA isolates showed 100 and 0% resistance to penicillin and vancomycin, respectively. The D-test showed inducible clindamycin-resistant phenotype in 15.4% of MRSA isolates. Conclusion:This demonstrates the utmost need for routine testing for MRSA in Nepalese hospitals.
Project description:Gene expression in human umbilical vein endothelial cells (HUVEC) was investigated by microarray analysis after 4 h infection with S. aureus isolated from healthy nasal carriers (n=5) and from blood (n=5) of septic patients. All bacterial isolates were spa-typed and characterized with a DNA microarray to determine the presence of virulence genes. Keywords: infection studies, pathogen, S. aureus Overall design: Five S. aureus (designated BI-BV) from a collection of blood culture isolates (Department of Clinical Microbiology, Ryhov County Hospital, Jönköping, Sweden) from septic patients were selected. Isolates from patients with diabetes, endocarditis, drug addicts and persons with an operation within the three last years were excluded. Two S. aureus isolates were from patients with an abscess in the psoas muscle, two from patients with spondylitis and one from a wound in the neck. Another five isolates (CI-CV) were randomly selected from a collection of S. aureus obtained from healthy male nasal carriers collected in a previous study.
Project description:Antibiotic resistant bacterial infections are a significant problem in the healthcare setting, in many cases requiring the rapid administration of appropriate and effective antibiotic therapy. Diagnostic assays capable of quickly and accurately determining the pathogen resistance profile are therefore crucial to initiate or modify care. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a standard method for species identification in many clinical microbiology laboratories and is well positioned to be applied towards antimicrobial susceptibility testing. One recently reported approach utilizes semi-quantitative MALDI-TOF MS for growth rate analysis to provide a resistance profile independent of resistance mechanism. This method was previously successfully applied to Gram-negative pathogens and mycobacteria; here, we evaluated this method with the Gram-positive pathogen Staphylococcus aureus. Specifically, we used 35 strains of S. aureus and four antibiotics to optimize and test the assay, resulting in an overall accuracy rate of 95%. Application of the optimized assay also successfully determined susceptibility from mock blood cultures, allowing both species identification and resistance determination for all four antibiotics within 3 hours of blood culture positivity.
Project description:Staphylococcus aureus, a notorious human pathogen, is a major cause of the community as well as healthcare associated infections. It can cause a diversity of recalcitrant infections mainly due to the acquisition of resistance to multiple drugs, its diverse range of virulence factors, and the ability to produce biofilm in indwelling medical devices. Such biofilm associated chronic infections often lead to increase in morbidity and mortality posing a high socio-economic burden, especially in developing countries. Since biofilm formation and antibiotic resistance function dependent on each other, detection of biofilm expression in clinical isolates would be advantageous in treatment decision. In this premise, we attempt to investigate the biofilm formation and its association with antibiotic resistance in clinical isolates from the patients visiting tertiary health care hospitals in Nepal. Bacterial cells isolated from clinical samples identified as S. aureus were examined for in-vitro biofilm production using both phenotypic and genotypic assays. The S. aureus isolates were also examined for susceptibility patterns of clinically relevant antibiotics as well as inducible clindamycin resistance using standard microbiological techniques and D-test, respectively. Among 161 S. aureus isolates, 131 (81.4%) were methicillin resistant S. aureus (MRSA) and 30 (18.6%) were methicillin sensitive S. aureus (MSSA) strains. Although a majority of MRSA strains (69.6%) showed inducible clindamycin resistance, almost all isolates (97% and 94%) were sensitive toward chloramphenicol and tetracycline, respectively. Detection of in vitro production of biofilm revealed the association of biofilm with methicillin as well as inducible clindamycin resistance among the clinical S. aureus isolates.
Project description:Macrolides, lincosamides, and type B streptogramins (MLS<sub>B</sub>) are important therapeutic options to treat methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infections; however, resistance to these antibiotics has been emerging. In Chile, data on the MLS<sub>B</sub> resistance phenotypes are scarce in both community-(CA) and hospital-acquired (HA) MRSA isolates. Antimicrobial susceptibility to MLS<sub>B</sub> was determined for sixty-eight non-repetitive isolates of each HA-(32) and CA-MRSA (36). Detection of SCC<i>mec</i> elements, <i>ermA</i>, <i>ermB</i>, <i>ermC</i>, and <i>msrA</i> genes was performed by PCR. The predominant clones were SCC<i>mec</i> I-ST5 (HA-MRSA) and type IVc-ST8 (CA-MRSA). Most of the HA-MRSA isolates (97%) showed resistance to clindamycin, erythromycin, azithromycin, and clarithromycin. Among CA-MRSA isolates, 28% were resistant to erythromycin, azithromycin, and 25% to clarithromycin. All isolates were susceptible to linezolid, vancomycin, daptomycin and trimethoprim/sulfamethoxazole, and over 97% to rifampicin. The <i>ermA</i> gene was amplified in 88% of HA-MRSA and 17% of CA-MRSA isolates (<i>p</i> < 0.001). The <i>ermC</i> gene was detected in 6% of HA-SARM and none of CA-SARM isolates, whereas the <i>msrA</i> gene was only amplified in 22% of CA-MRSA (<i>p</i> < 0.005). Our results demonstrate the prevalence of the cMLSB resistance phenotype in all HA-MRSA isolates in Chile, with the <i>ermA</i> being the predominant gene identified among these isolates.
Project description:Antibiotic resistance has been reported since the 1940s in both human and veterinary medicine. Many years of monitoring milk samples in South Africa led to identification of a novel maltose-negative Staphylococcus aureus (S. aureus) strain, which appears to be an emerging pathogen. In this study, the susceptibility of this strain to antibiotics was evaluated over time, during diverse seasons in various provinces and according to somatic cell count (SCC) categories. A data set of 271 maltose-negative S. aureus isolates, from milk samples of 117 dairy herds, was examined using the disk diffusion method, between 2010 and 2017. This study also compared the susceptibility testing of 57 maltose-negative and 57 maltose-positive S. aureus isolated from 38 farms, from three provinces using minimum inhibitory concentration (MIC). The MIC results for the maltose-negative S. aureus isolates showed highest resistance to ampicillin (100%) and penicillin (47.4) and lowest resistance (1.8%) to azithromycin, ciprofloxacin and erythromycin. The maltose-negative S. aureus isolates showed overall significantly increased antibiotic resistance compared to the maltose-positive strains, as well as multidrug resistance. Producers and veterinarians should consider probability of cure of such organisms (seemingly non-chronic) when adapting management and treatment, preventing unnecessary culling.
Project description:Staphylococcus aureus is an important human pathogen with an arsenal of virulence factors and a propensity to acquire antibiotic resistance genes. The understanding of the global epidemiology of S. aureus through the use of various typing methods is important in the detection and tracking of novel and epidemic clones in countries and regions. However, detailed information on antibiotic resistance and virulence genes of S. aureus, and its population structure is still limited in Africa. In this study, S. aureus isolates collected in South Africa (n = 38) and Nigeria (n = 2) from 2001-2004 were characterized by spa typing and DNA microarray. The combination of these two methods classified the isolates into seven spa types and three clonal complexes (CCs) i.e. t064-CC8 (n = 17), t037-CC8 (n = 8), t1257-CC8 (n = 6), t045-CC5 (n = 5), t951-CC8 (n = 1), t2723-CC88 (n = 1), t6238-CC8 (n = 1), and untypeable-CC8 (n = 1). A high percentage agreement (>95%) and kappa coefficient (>0.60) was largely observed with antibiotic susceptibility testing and DNA microarray, indicating substantial agreement. Some antibiotic and virulence gene markers were associated with specific clones. The detection of the collagen-binding adhesion (cna) gene was unique for t037-CC8-MRSA while the enterotoxin gene cluster (egc) and staphylococcal complement inhibitor (scn) gene were identified with t045-CC5-MRSA. Moreover, the combination of genes encoding enterotoxins (entA, entB, entK, entQ) was noted with most of the CC8 isolates. The t045-CC5-MRSA clone was positive for the mercury resistance (mer) operon. DNA microarray provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers for specific S. aureus clones in Africa.