Immunocompromised teenager with meningitis caused by Ureaplasma parvum.

Infection in the immunocompromised patient is often challenging on multiple levels. It can be difficult to distinguish between manifestations of the underlying disease, infection or malignancy. Symptoms may be vague or even absent, deviations in the common inflammatory parameters discrete, imaging findings scarce and the causative microbe may be a true pathogen as well as opportunistic. Here, we report an immunosuppressed female in her late teens with a purulent meningitis due to Ureaplasma parvum-a very rare cause of infection in the central nervous system of adults. We wish to highlight the relevance of intracellular pathogens and the need to actively search for these microbes, especially when response to broad-spectrum antibiotic treatment is absent. Furthermore, we emphasise the need for adequate molecular microbial diagnostics in search of microbes that are difficult to identify by culture and where serology and antigen tests may be absent or unreliable due to immune suppression.

Use of Sysmex UF-5000 flow cytometry in rapid diagnosis of urinary tract infection and the importance of validating carryover rates against bacterial count cut-off.

Introduction. Urinary tract infections are common bacterial infections worldwide. Urine culture is the gold standard method to identify and quantify the presence or absence of bacteria in urine. Flow cytometry, which can differentiate and quantify multiple particles (including bacteria) in the urine, presents an alternative method for rapid screening to rule out bacteriuria.Hypothesis. Adding flow cytometry to identify urine samples without bacteriuria could substantially reduce the number of urine samples that need to be cultured as well as the response time for negative results. However, the level of instrument rinsing between samples could affect sample-to-sample carryover rate, a concept given little attention in previous studies.Aim. We aimed to evaluate urine flow cytometry as a rapid screening method to identify urine samples without significant bacterial growth, including analyses of cross-contamination and sample-to-sample carryover rate.Methodology. We analysed 3919 urine samples by quantitative urine culture and flow cytometry screening (Sysmex UF-5000). Receiver operator characteristic (ROC) curve analyses were used to test method agreement to identify: (a) positive vs. negative culture and (b) mixed vs. pure culture. In addition, we performed carryover and cross-contamination studies.Results. ROC curve analyses identified bacterial count (BACT ml-1) and leucocyte count (WBC µl-1) as possible predictors of bacterial growth in the total material and subpopulations, except pregnant women (n=451). This subgroup was excluded from further analyses, leaving a final 3468 urine samples. Area under the ROC curve was 0.94 (95 % CI 0.93-0.95) and 0.81 (95 % CI 0.79-0.82) for bacterial and leucocyte count, respectively. A bacterial count cut-off of 30 BACT ml-1 resulted in 95.2 % sensitivity and 91.2 % negative predictive value, resulting in approximately 30 % of urine samples that could be reported as negative without culture. Use of high-level rinse modes was necessary to ensure carryover rates <0.05 %.Conclusion. Flow cytometry is a suitable and rapid method to rule out urine samples without significant bacterial growth. Rinses between samples should be adjusted, depending on the cut-off used, to prevent sample-to-sample carryover, whereas cross-contamination can be eliminated by the use of separate urine aliquots for flow cytometry analysis and urine culturing respectively.

Broad-Spectrum Antibacterial Peptide Kills Extracellular and Intracellular Bacteria Without Affecting Epithelialization.

New antibacterial drugs with novel modes of action are urgently needed as antibiotic resistance in bacteria is increasing and spreading throughout the world. In this study, we aimed to explore the possibility of using APIM-peptides targeting the bacterial ß-clamp for treatment of skin infections. We selected a lead peptide, named betatide, from five APIM-peptide candidates based on their antibacterial and antimutagenic activities in both G+ and G- bacteria. Betatide was further tested in minimal inhibitory concentration (MIC) assays in ESKAPE pathogens, in in vitro infection models, and in a resistance development assay. We found that betatide is a broad-range antibacterial which obliterated extracellular bacterial growth of methicillin-resistant Staphylococcus epidermidis (MRSE) in cell co-cultures without affecting the epithelialization of HaCaT keratinocytes. Betatide also reduced the number of intracellular Staphylococcus aureus in infected HaCaT cells. Furthermore, long-time exposure to betatide at sub-MICs induced minimal or no increase in resistance development compared to ciprofloxacin and gentamicin or ampicillin in S. aureus and Escherichia coli. These properties support the potential of betatide for the treatment of topical skin infections.

Counting Replication Origins to Measure Growth of Pathogens.

For the past several decades, the success of bacterial strains in infecting their host has been essentially ascribed to the presence of canonical virulence genes. While it is unclear how much growth rate impacts the outcome of an infection, it is long known that the efficacy of the most commonly used antibiotics is correlated to growth. This applies especially to -lactams, whose efficacy is nearly abolished when cells grow very slowly. It is therefore reasonable to assume that a niche or genetic dependent change in growth rate could contribute to the variability in the outcome of antibiotic therapy. However, little is known about the growth rate of pathogens or their pathotypes in their host.

Growth Rate of Escherichia coli During Human Urinary Tract Infection: Implications for Antibiotic Effect.

Escherichia coli is the primary cause of urinary tract infection (UTI), which is one of the most frequent human infections. While much is understood about the virulence factors utilized by uropathogenic E. coli (UPEC), less is known about the bacterial growth dynamics taking place during infection. Bacterial growth is considered essential for successful host colonization and infection, and most antibiotics in clinical use depend on active bacterial growth to exert their effect. However, a means to measure the in situ bacterial growth rate during infection has been lacking. Due to faithful coordination between chromosome replication and cell growth and division in E. coli, chromosome replication provides a quantitative measure of the bacterial growth rate. In this study, we explored the potential for inferring in situ bacterial growth rate from a single urine sample in patients with E. coli bacteriuria by differential genome quantification (ori:ter) performed by quantitative PCR. We found active bacterial growth in almost all samples. However, this occurs with day-to-day and inter-patient variability. Our observations indicate that chromosome replication provides not only a robust measure of bacterial growth rate, but it can also be used as a means to evaluate antibiotic effect.

Comparative Activity of Ceftriaxone, Ciprofloxacin, and Gentamicin as a Function of Bacterial Growth Rate Probed by Escherichia coli Chromosome Replication in the Mouse Peritonitis Model.

Commonly used antibiotics exert their effects predominantly on rapidly growing bacterial cells; yet, the growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, a means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated chromosome replication as a readout of in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (quantitative PCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average level and on a single-cell level, from one single biological specimen. Here, we asked whether the in situ growth rate predicts antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as a proof of concept. Our data demonstrate that the activities of the commonly used antibiotics ceftriaxone and gentamicin correlated with pretreatment bacterial growth rate; both drugs performed better during rapid growth than during slow growth. Conversely, ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotic's dependency on active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.

Chromosome replication as a measure of bacterial growth rate during Escherichia coli infection in the mouse peritonitis model.

The efficacy of most antibiotics is dependent on active bacterial growth, yet little is known about the growth dynamics during infection. Therefore, means to measure in-host bacterial growth rate is of importance. Here, we use chromosome replication as readout for in situ bacterial growth rate during infection; obtained from a single biological specimen. We have applied two independent methods: quantitative PCR (qPCR) and fluorescence microscopy, to quantify the level of chromosome replication present during Escherichia coli propagation in the mouse peritonitis model. We find that the methods complement each other and allow for quantification of growth rate, both on a population average and on a single-cell level. We demonstrate the presence of heterogeneous growth rates within bacterial populations propagating during infection. Also, no growth cessation was observed during the apparent stationary phase in vivo, and, by comparison of growth dynamics at different anatomical sites, we demonstrate that E. coli is unlikely to grow independently intravascularly. These findings provide novel insight into bacterial growth during host infection, and underscore the importance of pinpointing the primary site of infection in septicaemia of unknown origin and ensuring antibiotic availability at this site.