Real-time detection of blaKPC in clinical samples and surveillance specimens.

A real-time PCR assay was developed targeting the bla(KPC) responsible for Klebsiella pneumoniae carbapenemase (KPC)-mediated carbapenem resistance and was validated for testing colonies or enrichment broth cultures. The assay accurately detects KPC-containing strains with high analytical specificity and sensitivity.

Cytoskeletal Tropomyosin as a Biomarker in Clostridium difficile Infection.

BACKGROUND: Current diagnostics of Clostridium difficile infection (CDI) heavily relies on detection of the disease-causing organism. The objective of this study was to investigate a cytoskeletal protein, tropomyosin (Tpm), as a CDI biomarker. METHODS: Fecal Tpm was tested by monoclonal antibodies (mAbs) in a 12-month prospective study. Remnant diarrheal clinical specimens and relevant clinical data were collected. The CDI positive (CDI+, n = 230) and CDI negative (CDI-, n = 228) groups were composed of samples testing positive or negative by polymerase chain reaction (PCR) (Xpert® C. difficile/Epi, Cepheid), respectively. The other enteric pathogen (OEP) group (n = 52) was composed of specimens tested for the presence of other enteric pathogens or parasites by routine testing methods. Extracted fecal Tpm was detected by Western blot and the results were correlated with CDI based on clinical and microbiology laboratory data. RESULTS: A total of 510 stool specimens were tested. Tpm is not stable in stool, suggesting the utility of fresh specimens. In the CDI+ group, specificity and sensitivity of Tpm detection in correlation with a CDI were 93.2% and 53.7%, respectively, when only "true CDI" and "not CDI" were analyzed (110 samples). For CDI+ samples, 23% did not satisfy CDI clinical signs. Tpm positives in the CDI- group (8.3%) had inflammatory bowel diseases. CONCLUSION: Tpm has a potential role as a CDI biomarker in combination with C. difficile PCR and an appropriate clinical evaluation. However, non-muscle Tpm, as a biomarker for CDI, suffers from a low sensitivity in our study. Therefore further investigation using larger cohorts is needed.

Immunological Stability of Clostridium difficile Toxins in Clinical Specimens.

OBJECTIVE The impact of storage on stability and detection of Clostridium difficile toxins in feces is poorly understood. The objective of this study was to investigate the immunological stability of C. difficile toxins in clinical stool specimens under different storage conditions by evaluating this stability using toxin detection by enzyme immunoassay (EIA). METHODS Stool specimens positive for C. difficile infection (CDI) by quantitative polymerase chain reaction (qPCR) were used for EIA testing with the C. difficile Tox A/B II kit. The EIA-positive specimens were stored aerobically under refrigerated (4-10°C) and frozen (-30°C and -80°C) conditions. Measurement of toxin quantity was conducting using optical density (OD) on days 0, 14, 30, 60, 90, and 120 of storage. RESULTS Clostridium difficile toxins demonstrated good detection in undiluted stool specimens by EIA up to 120 days of storage. Good detection of the toxins was observed in diluted samples at refrigerated and -80°C temperatures. Dilution detrimentally affected toxin detection at -30°C. CONCLUSION Storage of undiluted clinical stool specimens at refrigerated, -30°C, and -80°C temperatures for up to 120 days has no discernible effect on the immunological stability of C. difficile cytotoxins. However, storage at -30°C has a detrimental effect on C. difficile toxin stability in diluted specimens. Infect Control Hosp Epidemiol 2018;39:434-438.

Host response to Clostridium difficile infection: Diagnostics and detection.

Clostridium difficile infection (CDI) is a significant healthcare concern worldwide, and C. difficile is recognised as the most frequent aetiological agent of infectious healthcare-associated diarrhoea in hospitalised adult patients. The clinical manifestation of CDI varies from self-limited diarrhoea to life-threatening colitis. Such a broad disease spectrum can be explained by the impact of host factors. Currently, a complex CDI aetiology is widely accepted, acknowledging the interaction between bacteria and the host. C. difficile strains producing clostridial toxins A and B are considered toxigenic and can cause disease; those not producing the toxins are non-pathogenic. A person colonised with a toxigenic strain will not necessarily develop CDI. It is imperative to recognise patients with active disease from those only colonised with this pathogen and to implement appropriate treatment. This can be achieved by diagnostics that rely on host factors specific to CDI. This review will focus on major aspects of CDI pathogenesis and molecular mechanisms, describing host factors in disease progression and assessment of the host response in order to facilitate the development of CDI-specific diagnostics.

Joint Transcriptional Control of Virulence and Resistance to Antibiotic and Environmental Stress in Acinetobacter baumannii.

UNLABELLED: The increasing emergence of antibiotic-resistant bacterial pathogens represents a serious risk to human health and the entire health care system. Many currently circulating strains of Acinetobacter baumannii exhibit resistance to multiple antibiotics. A key limitation in combating A. baumannii is that our understanding of the molecular mechanisms underlying the pathogenesis of A. baumannii is lacking. To identify potential virulence determinants of a contemporary multidrug-resistant isolate of A. baumannii, we used transposon insertion sequencing (TnSeq) of strain AB5075. A collection of 250,000 A. baumannii transposon mutants was analyzed for growth within Galleria mellonella larvae, an insect-based infection model. The screen identified 300 genes that were specifically required for survival and/or growth of A. baumannii inside G. mellonella larvae. These genes encompass both known, established virulence factors and several novel genes. Among these were more than 30 transcription factors required for growth in G. mellonella. A subset of the transcription factors was also found to be required for resistance to antibiotics and environmental stress. This work thus establishes a novel connection between virulence and resistance to both antibiotics and environmental stress in A. baumannii. IMPORTANCE: Acinetobacter baumannii is rapidly emerging as a significant human pathogen, largely because of disinfectant and antibiotic resistance, causing lethal infection in fragile hosts. Despite the increasing prevalence of infections with multidrug-resistant A. baumannii strains, little is known regarding not only the molecular mechanisms that allow A. baumannii to resist environmental stresses (i.e., antibiotics and disinfectants) but also how these pathogens survive within an infected host to cause disease. We employed a large-scale genetic screen to identify genes required for A. baumannii to survive and grow in an insect disease model. While we identified many known virulence factors harbored by A. baumannii, we also discovered many novel genes that likely play key roles in A. baumannii survival of exposure to antibiotics and other stress-inducing chemicals. These results suggest that selection for increased resistance to antibiotics and environmental stress may inadvertently select for increased virulence in A. baumannii.

Investigation of tigecycline bactericidal activity: Optimisation of laboratory testing.

The objectives of this study were to optimise the conditions for bactericidal testing of tigecycline and to investigate its bactericidal activity against clinical isolates of Gram-positive and Gram-negative bacteria. Tigecycline is the first in a new class of glycylcycline antibiotics exhibiting in vitro activity against a broad range of bacteria, including multidrug-resistant organisms. Its bactericidal activity in vitro has not been extensively investigated using multiple test conditions. Five growth media comprising Mueller Hinton broth, Minimum Essential Medium of Eagle, Ham F-12, RPMI 1640 and Iso-Sensitest broth (ISB) with and without surfactant (Tween 80) were investigated in vitro to assess tigecycline bactericidal activity. Clinical isolates of meticillin-resistant Staphylococcus aureus, meticillin-susceptible S. aureus, Escherichia coli, Klebsiella pneumoniae and Enterococcus spp., representing the majority of clinically relevant bacteria, were evaluated for the impact of test conditions on the tigecycline minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), ISB with 0.02% Tween 80 most efficiently demonstrated the bactericidal action of tigecycline when evaluated in 64 well-characterised clinical isolates and was considered as the optimal bactericidal test medium. Using this condition, tigecycline approached 56% bactericidal activity with 3log10 reduction in CFUs at 72h incubation. Bactericidal action increased to 80% of strains when 2log10 reduction was used as the endpoint. Only Enterococcus spp. showed no bactericidal response in this analysis. Tigecycline exhibited a bactericidal effect in vitro against Gram-positive and Gram-negative bacteria. At the tested in vitro conditions, tigecycline MICs were unchanged regardless of the different test media used.

Prospective, multicenter evaluation of the BD GeneOhm VanR assay for direct, rapid detection of vancomycin-resistant Enterococcus species in perianal and rectal specimens.

The BD GeneOhm VanR assay (BD Diagnostics, San Diego, CA), a qualitative test for the rapid detection of vancomycin-resistant enterococci (VRE) from rectal and/or perianal swabs, combines integrated nucleic acid extraction and automated polymerase chain reaction for the detection of vanA and/or vanB gene sequences. We studied 1,027 perianal and rectal swab specimens from 3 geographically distinct US sites (prevalence rates, 13.1%-25.8%). Direct swab specimens were tested by the assay and compared with direct culture. The sensitivity, specificity, and positive and negative predictive values of the assay were 93.2%, 81.9%, 54.4%, and 98.1%, respectively. The specificity was limited largely due to false-positives in the vanB portion of the assay. Specificity with perianal swabs was significantly greater than with rectal swabs, 87.1% vs 74.7%, respectively (P < .0001). When used only to detect resistance conferred by vanA, the assay was 88.3% (158/179) sensitive and 95.8% (802/837) specific, with positive and negative predictive values of 81.9% and 97.4%, respectively. The assay is a simple, rapid, and acceptable method for screening for VRE in a variety of populations in which vanA is the predominant genotype. Samples positive for the vanB genotype should be confirmed by culture owing to the apparent high number of false-positive results.