Peter Jelfs

Outbreak of extended spectrum beta lactamase producing Klebsiella pneumoniae in a neonatal unit.

An outbreak of extended spectrum β lactamase producingKlebsiella pneumoniae (ESBLKp) in a neonatal unit was controlled using simple measures. Normally, the control of such infections can be time consuming and expensive. Seven cases of septicaemia resulted in two deaths. ESBLKp isolates were subtyped by pulsed field gel electrophoresis, and four of the five isolates typed were identical. Control of the outbreak was achieved by altered empiric antibiotic treatment for late onset sepsis and prevention of cross infection by strict attention to hand washing. Widespread colonisation of babies in the unit was presumed, so initial surveillance cultures were not performed. No further episodes of sepsis occurred.

Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

ABSTRACT We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

MUTATIONS IN rpoB GENE AND RIFABUTIN SUSCEPTIBILITY OF MULTIDRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS ISOLATED IN AUSTRALIA

Control of tuberculosis, the single largest killer among the infectious diseases, has been threatened by the emergence of multidrug-resistant Mycobacterium tuberculosis (MDRTB) infection due to the limited treatment options. Rifampicin (RIF) resistance is considered as a marker for MDRTB. The aim of this study was the detection of rpoB gene mutations and rifabutin resistance in MDRTB strains recently isolated in Australia by a line probe assay (INNO-LiPA Rif. TB, Innogenetics). Rifabutin and RIF susceptibility of 20 MDRTB and 16 RIF-sensitive M. tuberculosis complex clinical isolates were studied. The overall concordance of the line probe assay (LiPA) with phenotypic RIF susceptibility test was 96%. Seven distinct nucleotide substitutions were identified in 21 of 22 RIF-resistant isolates of diverse geographical origins, but in none of the RIF-sensitive strains. The majority (71%) of mutations occurred in the 526-533 codons and were associated with resistance to rifabutin and RIF. Of the RIF-resistant MDRTB strains, 18% appeared to be rifabutin-sensitive and produced delta S2 and delta S3 INNO-LiPA patterns. We conclude that amino acid substitutions at Asp516 and Ser522 in the rpoB gene in RIF-resistant M. tuberculosis predict rifabutin susceptibility for MDRTB. Use of the LiPA for RIF and rifabutin resistance may facilitate the rapid response required to limit the extent and severity of MDRTB transmission and infection.

Recurrence of tuberculosis in a low-incidence setting

Recurrence of active tuberculosis following treatment of an initial disease episode can occur due to endogenous re-activation or exogenous re-infection. Cases of recurrent tuberculosis in the Australian state of New South Wales between 1994 and 2006 were identified by data linkage analysis with confirmatory review of case notes. Patients with more than one culture-positive disease episode during that time period who had completed treatment for the initial disease episode were included. Genotyping of Mycobacterium tuberculosis was used to determine whether recurrence was likely to be due to re-activation or re-infection. There were 5,723 tuberculosis notifications between 1994 and 2006, 3,731 of which were culture-positive. Fifteen (0.4%) patients had recurrent culture-positive disease over a mean 5.7 yrs of follow-up (crude annual incidence 71 per 100,000 population). Recurrent tuberculosis was attributable to re-activation (indistinguishable strains) in 11 (73%) cases and to re-infection (different strains) in four (27%). In a low-incidence setting of tuberculosis, a control programme incorporating directly observed therapy for active disease resulted in a very low rate of recurrent tuberculosis over a long period of follow-up. Re-infection is less likely than re-activation, but still contributes significantly to the number of cases with recurrent disease.

Added value of whole-genome sequencing for management of highly drug-resistant TB

Objectives Phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis takes several weeks to complete and second-line DST is often poorly reproducible, potentially leading to compromised clinical decisions. Following a fatal case of XDR TB, we investigated the potential benefit of using whole-genome sequencing to generate an in silico drug susceptibility profile. Methods The clinical course of the patient was reviewed, assessing the times at which phenotypic DST data became available and changes made to the therapeutic regimen. Whole-genome sequencing was performed on the earliest available isolate and variants associated with drug resistance were identified. Results The final DST report, including second-line drugs, was issued 10 weeks after patient presentation and 8 weeks after initial growth of M. tuberculosis. In the interim, the patient may have received a compromised regimen that had the potential to select for further drug resistance. The in silico susceptibility profile, extrapolated from evolving evidence in the literature, provided comparable or superior data to the DST results for second-line drugs and could be generated in a much shorter timeframe. Conclusions We propose routine whole-genome sequencing of all MDR M. tuberculosis isolates in adequately resourced settings. This will improve individual patient care, monitor for transmission events and advance our understanding of resistance-associated mutations.

Phenotypically occult multidrug-resistant Mycobacterium tuberculosis: dilemmas in diagnosis and treatment

OBJECTIVES The clinical significance of the emergence of Mycobacterium tuberculosis (MTB) isolates that contain rpoB mutations (genotypic resistance), but are phenotypically susceptible to rifampicin (RIF G(R) P(S)), remains uncertain. The aim of this study was to determine the prevalence of MTB cases that demonstrate this discordant rifampicin resistance pattern and to establish whether these patients have poorer treatment outcomes with rifampicin-based regimens. METHODS rpoB sequencing was performed on all MTB isolates demonstrating phenotypic resistance to one or more first-line antituberculosis agents (excluding rifampicin). Rifampicin MICs were determined for rpoB mutation-positive isolates and clinical case notes were reviewed to identify treatment outcomes in these patients. RESULTS Of the 214 phenotypically drug (excluding rifampicin)-resistant isolates tested, 5 contained rpoB mutations (4 isoniazid resistant and 1 pyrazinamide resistant). These isolates demonstrated elevated rifampicin MICs (low-level resistance), despite testing susceptible using phenotypic broth-based methods. One patient experienced a relapse of tuberculosis (TB) 2 years after completion of a rifampicin-containing regimen. These findings are consistent with a recent study that reported treatment failure with rifampicin-based regimens in patients with isoniazid-resistant MTB and genotypic rifampicin resistance. CONCLUSIONS While MTB RIF G(R) P(S) strains remain relatively uncommon, they can be associated with low-level rifampicin resistance and poorer treatment outcomes with rifampicin-based regimens. This recently recognized form of multidrug-resistant TB should be adequately detected and managed.

Difficulties in detection and identification of Enterococcus faecium with low-level inducible resistance to vancomycin, during a hospital outbreak

Between June and November 2004, a vancomycin-resistant Enterococcus faecium (VRE) strain was isolated from 13 patients in the haematology/bone marrow transplant unit. There were difficulties in identifying the organism, which had low-level, inducible vancomycin resistance, and standard screening methods did not reveal carriage in patients or their contacts. These technical failures led to spread of VRE and delays in providing appropriate management, which might otherwise have been avoided. Therefore, we reviewed our laboratory methods and compared three identification systems to determine which would best identify this VRE strain. The VITEK 2 (BioMerieux) correctly identified, as E. faecium, only two of 16 isolates, whereas API Rapid ID 32 Strep (BioMerieux) and Phoenix 100 (Becton Dickinson and Co.) correctly identified 13 of 15 and 12 of 13 isolates tested, respectively. Isolates from urine, tested by the CLSI disk diffusion method, were apparently susceptible or of intermediate susceptibility to vancomycin, upon primary testing. VITEK 2 and Phoenix 100 identified all isolates as vancomycin-resistant, although the MICs, measured by Etest, were in the susceptible range for three of 16 isolates. Reducing the vancomycin concentration in screening media substantially increased the sensitivity for detection of VRE. Isolates were characterized as genotype vanB2/3 by PCR and were indistinguishable from each other by pulsed-field gel electrophoresis. VRE with low-level inducible resistance can be missed by routine screening methods. Better identification and screening methods for detection of low-level vancomycin resistance are needed to improve surveillance and prevent transmission of VRE.

Extended phage locus typing of Salmonella enterica serovar Typhimurium, using multiplex PCR-based reverse line blot hybridization.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is the commonest pathogen causing food-borne disease among humans and animals in Australia. A multiplex PCR-based reverse line blot (mPCR/RLB) system was developed to rapidly identify S. Typhimurium phage types and strains within them. The system comprised 32 biotin-labelled primer sets and 38 amino-labelled probes, based on sequences that were either phage-type-related or derived from temperate phages ST64B, P22, Gifsy-1 or Gifsy-2. The system was developed and evaluated using 168 S. Typhimurium isolates, representing 46 phage types. RLB patterns, based on a combination of positive hybridization and grading of signal intensities, validated by sequencing, differentiated S. Typhimurium isolates into 102 types. Some clusters contained isolates belonging to a single phage type while others contained isolates belonging to more than one. Most phage types exhibited at least two RLB profiles. The feasibility of this system was evaluated during investigations of three outbreaks, due to two different phage types. Within each outbreak, isolates showed identical RLB patterns, whereas sporadic isolates of corresponding phage types showed various patterns. The mPCR/RLB system was compared with multilocus variable-number tandem-repeat analysis (MLVA). The two methods demonstrated similar discriminatory abilities. Based on these preliminary results, the mPCR/RLB system is a promising tool for molecular identification of most common S. Typhimurium phage types. It could be used as an alternative to, or in conjunction with, MLVA for rapid strain typing during outbreaks.

Potent Antimycobacterial Activity of the Pyridoxal Isonicotinoyl Hydrazone Analog 2-Pyridylcarboxaldehyde Isonicotinoyl Hydrazone: A Lipophilic Transport Vehicle for Isonicotinic Acid Hydrazide

The rise in drug-resistant strains of Mycobacterium tuberculosis is a major threat to human health and highlights the need for new therapeutic strategies. In this study, we have assessed whether high-affinity iron chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class can restrict the growth of clinically significant mycobacteria. Screening a library of PIH derivatives revealed that one compound, namely, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), exhibited nanomolar in vitro activity against Mycobacterium bovis bacille Calmette-Guérin and virulent M. tuberculosis. Interestingly, PCIH is derived from the condensation of 2-pyridylcarboxaldehyde with the first-line antituberculosis drug isoniazid [i.e., isonicotinic acid hydrazide (INH)]. PCIH displayed minimal host cell toxicity and was effective at inhibiting growth of M. tuberculosis within cultured macrophages and also in vivo in mice. Further, PCIH restricted mycobacterial growth at high bacterial loads in culture, a property not observed with INH, which shares the isonicotinoyl hydrazide moiety with PCIH. When tested against Mycobacterium avium, PCIH was more effective than INH at inhibiting bacterial growth in broth culture and in macrophages, and also reduced bacterial loads in vivo. Complexation of PCIH with iron decreased its effectiveness, suggesting that iron chelation may play some role in its antimycobacterial efficacy. However, this could not totally account for its potent efficacy, and structure-activity relationship studies suggest that PCIH acts as a lipophilic vehicle for the transport of its intact INH moiety into the mammalian cell and the mycobacterium. These results demonstrate that iron-chelating agents such as PCIH may be of benefit in the treatment and control of mycobacterial infection.

Three-year longitudinal study of genotypes of Mycobacterium tuberculosis in a low prevalence population

Objective: To investigate the molecular epidemiology of tuberculosis, temporal and spatial distribution of Mycobacterium tuberculosis isolates and associations between genotypes and clinical characteristics, in a low prevalence population. Methods: A total of 930 M. tuberculosis isolates referred to the New South Wales (NSW, Australia) Mycobacterium Reference Laboratory in 2004–2006 were characterised by mycobacterial interspersed repetitive unit (MIRU) and spacer oligonucleotide (spoligo) typing. Associations between genotypes, patient age, disease site and drug resistance were explored and the predictive power of molecular typing was analysed using Bayesian Belief Networks. Results: Among isolates from 855 NSW residents, there were 287 spoligotypes, 494 MIRU types and 643 unique spoligotype‐MIRU type combinations. They formed 73 spoligotype, 104 MIRU type and 76 spoligo‐MIRU clusters, most of which contained only two isolates. The majority (87.7%) of spoligotype clusters contained several MIRU profiles and 64.4% of MIRU clusters contained several spoligotypes. The three most common M. tuberculosis clades were Beijing (24.1%), East African Indian (11.8%) and Central Asian (6.5%); 6.9% and 0.7% isolates were resistant to isoniazid and rifampicin, respectively. There was no proof of association between genotype and drug resistance but isoniazid resistance increased independently over time. Given the low rates of genotype clustering, statistical analysis of genotype‐phenotype associations was limited. Potential associations were not confirmed by Bayesian classifiers. Conclusions: Spoligo and MIRU typing demonstrated low levels of M. tuberculosis clustering in NSW; temporal and spatial changes in M. tuberculosis genotypes reflected migration patterns to Australia. No analytically significant associations between M. tuberculosis genotypes and clinical phenotypes were detected.