Understanding vancomycin tolerance in hospital-acquired methicillin-resistant Staphylococcus aureus isolates from patients with sustained bacteraemia

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections including bacteraemia. Vancomycin has traditionally been the antibiotic of choice for treatment, but despite using the minimum inhibitory concentration (MIC) to guide treatment management, some patients still fail antibiotic therapy even when infected with strains that test as susceptible. This is further exacerbated with the emergence of vancomycin intermediate S. aureus (VISA) and heterogeneous vancomycin intermediate S. aureus (hVISA) strains, which exhibit reduced susceptibility to vancomycin. When exposed to sub-lethal concentrations of vancomycin, these VISA and hVISA subtypes are believed to undergo cell wall metabolism changes which increases the thickness of their cell walls and this form of resistance has been associated with sustained bacteraemia and increased mortality. Antibiotic tolerance is a phenotypic trait where an organism is resistant to the lethal killing of an antibiotic despite being inhibited by normal concentrations, and may explain the poorer outcome seen in patients with sustained bacteraemia. In vitro this interaction between an antibiotic concentration and bacterial growth or killing can be measured by the MIC (antibiotic concentration required to inhibit bacterial growth) and the minimum bactericidal concentration (MBC; that antibiotic concentration required to kill the bacterium) respectively. Using these two measures, tolerance is defined when the ratio of an isolates measured MBC to MIC is >32 after 24 hrs of incubation. Despite guidelines for MBC testing being issued in 1999, there is ongoing variation in the methodology used among testing laboratories, and the disparity in reported tolerance rates among S. aureus strains has lead clinicians to question the utility of MBC testing. Although there is conflicting data on the clinical significance of vancomycin-tolerant staphylococci, evidence suggests that tolerance may be an independent risk factor for poorer outcome, and the close association with hVISA and VISA strains may provide an understanding of the mechanisms that drive vancomycin resistance. This study investigated whether variations in the methodology used in MBC and time-kill tests impacted the ability to detect antibiotic tolerance. This was achieved determining the effect of using different media and test conditions on tolerance rates seen in a collection of hVISA and VISA isolates from an ST239 dominant MRSA population. The results from this study demonstrate that MBC results vary between tests and that the detection of antibiotic tolerance is highly dependent on testing conditions. Time-kill assays are the recommended method for detecting tolerance, and if MBC testing were to be performed, then it should be performed after isolates have undergone an in vitro vancomycin pre-exposure step. To better understand antibiotic tolerance and the mechanisms that drive vancomycin reduced susceptibility, MBC testing must be better standardised, and furthermore, the findings from this thesis propose an enhancement to current testing methodology which can be used in future large-scale studies to determine the clinical relevance of antibiotic tolerance

The Small Colony Variant of<i> Listeria monocytogenes</i> Is More Tolerant to Antibiotics and Has Altered Survival in RAW 264.7 Murine Macrophages

Small Colony Variant (SCV) cells of bacteria are a slow-growing phenotype that result from specific defects in the electron transport chain. They form pinpoint colonies on agar plates and have a variety of phenotypic characteristics, such as altered carbon metabolism, decreased toxin and lytic enzyme production, aminoglycoside resistance, and increased intracellular persistence. They are clinically relevant in Staphylococcus aureus and Pseudomonas aeruginosa, serving as a reservoir for recurrent or prolonged infections. Here, we found that a SCV mutant in the foodborne pathogen Listeria monocytogenes (strain SCV E18), similar to the high persister mutant phenotype, survived significantly better than the wild type when exposed over a 48-h period to concentrations above Minimal Inhibitory Concentration for most tested antibiotics. SCV E18 survived more poorly than the wildtype in unactivated RAW264.7 macrophage cells, presumably because of its reduced listeriolysin O expression, however, it survived better in reactive oxygen species producing, phorbol 12-myristate 13-acetate-activated macrophages. Although SCV E18 was sensitive to oxygen as it entered the stationary phase, it was significantly more tolerant to H(2)O(2) than the wild type, which may result from a shift in metabolism, however, further investigation is needed to resolve this. SCV E18 is a spontaneous mutant with a point mutation in the hemA gene. A wild type copy of hemA was complemented on plasmid pSOG30222, which restored the wild type phenotype. The results reported here suggest that the SCV of L. monocytogenes could be of clinical importance and highlight a need for adequate clinical screening for this phenotype, as it could affect antibiotic treatment outcomes

“Tolerance” of Misused Terminology? Enforcing Standardized Phenotypic Definitions

The recent paper by Haaber and colleagues entitled “Reversible Antibiotic Tolerance Induced in Staphylococcus aureus by Concurrent Drug Exposure” (1) revealed a possible alternative mechanism by which pathogens become less susceptible to standard therapy by screening for inducible antibiotic resistance in Staphylococcus aureus USA300 strain FP3757. We agree with the sentiments expressed by Bean and Wigmore (2) about the timeliness of this article and the need to examine antibiotic combinations, especially with increasing multidrug-resistant pathogens. Furthermore, this article highlighted some of the potential pitfalls of combination therapy and stressed the need for further research in this area. However, we have some concerns regarding the terminology and methods used in this study

The use of whole-genome sequencing for molecular epidemiology and antimicrobial surveillance : identifying the role of IncX3 plasmids and the spread of blaNDM-4-like genes in the Enterobacteriaceae

Aims. To characterise the resistome of a multi-drug resistant Klebsiella pneumoniae (Kp0003) isolated from an Australian traveller who was repatriated to a Sydney Metropolitan Hospital from Myanmar with possible prosthetic aortic valve infective endocarditis. Methods. Kp0003 was recovered from a blood culture of the patient and whole genome sequencing was performed. Read mapping and de novo assembly of reads facilitated in silico multi-locus sequence and plasmid replicon typing as well as the characterisation of antibiotic resistance genes and their genetic context. Conjugation experiments were also performed to assess the plasmid (and resistance gene) transferability and the effect on the antibiotic resistance phenotype. Results. Importantly, and of particular concern, the carbapenem-hydrolysing β-lactamase gene blaNDM-4 was identified on a conjugative IncX3 plasmid (pJEG027). In this respect, the blaNDM-4 genetic context is similar (at least to some extent) to what has previously been identified for blaNDM-1 and blaNDM-4-like variants. Conclusions. This study highlights the potential role that IncX3 plasmids have played in the emergence and dissemination of blaNDM-4-like variants worldwide and emphasises the importance of resistance gene surveillance

"Tolerance” of misused terminology? : enforcing standardized phenotypic definitions

The recent paper by Haaber and colleagues entitled “Reversible Antibiotic Tolerance Induced in Staphylococcus aureus by Concurrent Drug Exposure” (1) revealed a possible alternative mechanism by which pathogens become less susceptible to standard therapy by screening for inducible antibiotic resistance in Staphylococcus aureus USA300 strain FP3757. We agree with the sentiments expressed by Bean and Wigmore (2) about the timeliness of this article and the need to examine antibiotic combinations, especially with increasing multidrug-resistant pathogens. Furthermore, this article highlighted some of the potential pitfalls of combination therapy and stressed the need for further research in this area. However, we have some concerns regarding the terminology and methods used in this study