Emergence of OXA-48 and OXA-181 carbapenemases among Enterobacteriaceae in South Africa and evidence of in vivo selection of colistin resistance as a consequence of selective decontamination of the gastrointestinal tract.

This study reports on the emergence of OXA-48-like carbapenemases among isolates of Enterobacteriaceae in South Africa. In addition, the emergence during therapy of a colistin-resistant OXA-181-producing Klebsiella pneumoniae isolate was documented following selective digestive tract decontamination with oral colistin, which is therefore strongly discouraged.

Xpert MTB/RIF for rapid diagnosis of tuberculous lymphadenitis from fine-needle-aspiration biopsy specimens.

This study demonstrates the excellent diagnostic accuracy of the Xpert MTB/RIF test in patients with tuberculous lymphadenitis. The test sensitivity and specificity were 96.7% (95% confidence interval [CI], 86.6 to 100%) and 88.9% (95% CI, 69.6 to 100%), respectively, and it correctly identified 6/6 (100%) of the cytology smear-negative/culture-positive cases and 1 of 2 (50%) rifampin-resistant cases.

I-PASS Illness Severity Identifies Patients at Risk for Overnight Clinical Deterioration.

BACKGROUND: The I-PASS framework is increasingly being adopted for patient handoffs after a recent study reported a decrease in medical errors and preventable adverse events. A key component of the I-PASS handoff included assignment of illness severity. OBJECTIVE: We evaluated whether illness severity categories can identify patients at higher risk of overnight clinical deterioration as defined by activation of the rapid response team (RRT). METHODS: The I-PASS handoff documentation created by internal medicine residents and patient charts with overnight RRT activations from April 2016 through March 2017 were reviewed retrospectively. The RRT activations, illness severity categories, vital signs prior to resident handoff, and patient outcomes were evaluated. RESULTS: Of the 28 235 written patient handoffs reviewed, 1.3% were categorized as star (sickest patients at risk for higher level of care), 18.8% as watcher (unsure of illness trajectory), and 79.9% as stable (improving clinical status). Of the 98 RRT activations meeting the inclusion criteria, 5.1% were labeled as star, 35.7% as watcher, and 59.2% as stable. Patients listed as watcher had an odds ratio of 2.6 (95% confidence interval 1.7-3.9), and patients listed as star had an odds ratio of 5.2 (95% confidence interval 2.1-13.1) of an overnight RRT activation compared with patients listed as stable. The overall in-hospital mortality of patients with an overnight RRT was 29.6%. CONCLUSIONS: The illness severity component of the I-PASS handoff can identify patients at higher risk of overnight clinical deterioration and has the potential to help the overnight residents prioritize patient care.

Differential RD-1-specific IFN-γ host responses to diverse Mycobacterium tuberculosis strains in HIV-uninfected persons may be explained by genotypic variation in the ESX-1 region.

OBJECTIVES: Between-person variability in T-cell-specific interferon-gamma release assay (IGRA) responses and discordance between IGRA test formats are poorly understood. METHODS: We evaluated the IFN-γ responses (QuantiFERON-TB Gold-In-Tube [QFT-GIT] and TSPOT-TB) stratified according to the Mycobacterium tuberculosis spoligotype of the culture isolate obtained from the same patients with confirmed active tuberculosis (n = 91). We further analysed differences within the RD-1-encoding ESX-1 region between the different strain types using whole genome sequencing. RESULTS: In HIV-uninfected patients, TSPOT.TB and QFT-GIT IFN-γ responses were 5-fold (p < 0.01) and 2-fold higher (p < 0.05) for those infected with family 33 compared to the LAM strain (additionally, TSPOT.TB responses were 5.6-fold [p < 0.05] and 2.6-fold higher [p < 0.05] for the patients infected with the family 33 versus the X strain and Beijing versus the LAM strain, respectively). Multivariate analysis revealed that strain type (determined by spoligotyping) was independently associated with the magnitude of the IGRA response (varied by IGRA test type) and this is likely explained by variability in the ESX-1 region of Mycobacteriumtuberculosis (determined by next-generation sequencing). CONCLUSIONS: These data have implications for the understanding of between-person heterogeneity in IGRA responses, Mycobateriumtuberculosis-specific host immunity, and the discordance between different IGRA test formats.

Comparison of Neisseria gonorrhoeae minimum inhibitory concentrations obtained using agar dilution versus microbroth dilution methods.

With increasing antibiotic resistance observed amongst clinical isolates of Neisseria gonorrhoeae, the second most prevalent sexually transmitted bacterial disease in the United States, there is still a need for antimicrobial susceptibility testing (AST). The current method recommended by the Clinical and Laboratory Standards Institute is agar dilution. In this study, we show that a commercially available version of Fastidious Broth is capable of supporting N. gonorrhoeae in the evaluation of minimum inhibitory concentrations of 4 antibiotics (ceftriaxone, azithromycin, ciprofloxacin, and tetracycline), when comparing the agar dilution (AD) versus microbroth dilution (MBD) method and the susceptibilities obtained for 32 N. gonorrhoeae isolates. Herein, 3 out of the 4 antibiotics tested showed 94% or greater essential agreement (EA) and 91% or greater categorical agreement (CA) respectively, when comparing the MBD versus AD methods.

Seeing red; the development of pON.mCherry, a broad-host range constitutive expression plasmid for Gram-negative bacteria.

The development of plasmid-mediated gene expression control in bacteria revolutionized the field of bacteriology. Many of these expression control systems rely on the addition of small molecules, generally metabolites or non-metabolized analogs thereof, to the growth medium to induce expression of the genes of interest. The paradigmatic example of an expression control system is the lac system from Escherichia coli, which typically relies on the Ptac promoter and the Lac repressor, LacI. In many cases, however, constitutive gene expression is desired, and other experimental approaches require the coordinated control of multiple genes. While multiple systems have been developed for use in E. coli and its close relatives, the utility and/or functionality of these tools does not always translate to other species. For example, for the Gram-negative pathogen, Legionella pneumophila, a causative agent of Legionnaires' Disease, the aforementioned Ptac system represents the only well-established expression control system. In order to enhance the tools available to study bacterial gene expression in L. pneumophila, we developed a plasmid, pON.mCherry, which confers constitutive gene expression from a mutagenized LacI binding site. We demonstrate that pON.mCherry neither interferes with other plasmids harboring an intact LacI-Ptac expression system nor alters the growth of Legionella species during intracellular growth. Furthermore, the broad-host range plasmid backbone of pON.mCherry allows constitutive gene expression in a wide variety of Gram-negative bacterial species, making pON.mCherry a useful tool for the greater research community.

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.

Molecular characterisation and epidemiological investigation of an outbreak of blaOXA-181 carbapenemase-producing isolates of Klebsiella pneumoniae in South Africa.

BACKGROUND: Klebsiella pneumoniae is an opportunistic pathogen often associated with nosocomial infections. A suspected outbreak of K. pneumoniae isolates, exhibiting reduced susceptibility to carbapenem antibiotics, was detected during the month of May 2012 among patients admitted to a haematology unit of a tertiary academic hospital in Cape Town, South Africa (SA). OBJECTIVES: An investigation was done to determine possible epidemiological links between the case patients and to describe the mechanisms of carbapenem resistance of these bacterial isolates. METHODS: Relevant demographic, clinical and laboratory information was extracted from hospital records and an observational review of infection prevention and control practices in the affected unit was performed. Antimicrobial susceptibility testing including phenotypic testing and genotypic detection of the most commonly described carbapenemase genes was done. The phylogenetic relationship of all isolates containing the blaOXA-181 carbapenemase gene was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. RESULTS: Polymerase chain reaction analysis identified a total of seven blaOXA-181-positive, carbapenem-resistant K. pneumoniae isolates obtained from seven patients, all from a single unit. These isolates were indistinguishable using PFGE analysis and belonged to sequence type ST-14. No other carbapenemase enzymes were detected. CONCLUSION: This is the first documented laboratory-confirmed outbreak of OXA-181-producing K. pneumoniae in SA, and highlights the importance of enforcing strict adherence to infection control procedures and the need for ongoing surveillance of antibiotic-resistant pathogens in local hospitals.

Rapid diagnosis of pediatric mycobacterial lymphadenitis using fine needle aspiration biopsy.

BACKGROUND: Diagnosis of tuberculosis in children is challenging and fine needle aspiration biopsy (FNAB) is used worldwide in the diagnosis of palpable masses including peripheral lymphadenopathy. Recent studies of the use of nucleic acid amplification such as the Xpert MTB/RIF test on FNAB in adult patients have shown considerable promise. Xpert MTB/RIF allows for the rapid diagnosis of Mycobacterium tuberculosis and identification of rifampicin susceptibility. Studies to date have been predominantly performed in adults. This study aims to determine the accuracy of Xpert MTB/RIF for the detection of M. tuberculosis complex in FNAB from children with clinically suspected mycobacterial lymphadenitis. METHODS: Prospective hospital-based study of children <13 years referred for FNAB at Tygerberg hospital and Dora Nginza hospital, South Africa, for suspected mycobacterial lymphadenitis. Aspirates were performed and the results of the Xpert MTB/RIF test were compared with liquid (mycobacterial growth indicator tube) culture and cytology. RESULTS: FNABs were collected from 110 children and 38 (35%) cases were excluded. Of the 72 cases included in the study, 32 were positive for M. tuberculosis complex on Xpert MTB/RIF, 36 on cytology and 25 were culture positive for M. tuberculosis complex. Compared with the combined reference standard (cytomorphology suggestive of mycobacterial disease with direct visualization of the organism and/or bacteriological culture), Xpert MTB/RIF identified 32 of 40 cases as positive with a sensitivity and a specificity of 80% and 93.8%, respectively. CONCLUSIONS: FNAB and Xpert MTB/RIF enable a rapid diagnosis in pediatric mycobacterial lymphadenitis, expediting appropriate treatment and potentially preventing morbidity and mortality.

Outbreak of multi-drug resistant Pseudomonas aeruginosa bloodstream infection in the haematology unit of a South African Academic Hospital.

OBJECTIVE: To describe an outbreak of multi-resistant Pseudomonas aeruginosa bloodstream infections (MRPA-BSI) that occurred in the haematology ward of a tertiary academic hospital in Cape Town, South Africa, and determine risk factors for acquisition of MRPA-BSI. METHODS: The outbreak investigation included a search for additional cases, review of patient records, environmental and staff screening, molecular typing using pulsed-field gel electrophoresis (PFGE) and Multi-locus sequencing (MLST) and a retrospective case-control study. RESULTS: Ten MRPA-BSI cases occurred in the haematology ward between January 2010 and January 2011. The case fatality rate was 80%. Staff screening specimens were negative for MRPA and an environmental source was not identified. PFGE showed that 9/10 isolates were related. MLST showed that 3 of these 9 isolates belonged to Sequence type (ST) 233 while the unrelated isolate belonged to ST260. CONCLUSION: We have described an outbreak of MRPA-BSI occurring over an extended period of time among neutropenic haematology patients. Molecular typing confirms that the outbreak was predominantly due to a single strain. The source of the outbreak was not identified, but the outbreak appears to have been controlled following intensive infection control measures.