Jacqueline Schooneveldt

Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry To Identify Vancomycin-Resistant Enterococci and Investigate the Epidemiology of an Outbreak

ABSTRACT The control of vancomycin-resistant enterococci (VRE) has become an increasing burden on health care resources since their discovery over 20 years ago. Current techniques employed for their detection include time-consuming and laborious phenotypic methods or molecular methods requiring costly equipment and consumables and highly trained staff. An accurate, rapid diagnostic test has the ability to greatly reduce the spread of this organism, which has the ability to colonize patients for long periods, potentially even lifelong. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a technology with the ability to identify organisms in seconds and has shown promise in the identification of other forms of antimicrobial resistance in other organisms. Here we show that MALDI-TOF MS is capable of rapidly and accurately identifying vanB-positive Enterococcus faecium VRE from susceptible isolates. Internal validation of the optimal model generated produced a sensitivity of 92.4% and a specificity of 85.2%. Prospective validation results, following incorporation into the routine laboratory work flow, demonstrated a greater sensitivity and specificity at 96.7% and 98.1%, respectively. In addition, the utilization of MALDI-TOF MS to determine the relatedness of isolates contributing to an outbreak is also demonstrated.

Staphylococcus aureus Genotyping Using Novel Real-Time PCR Formats

ABSTRACT One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpsons index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of “light upon extension” (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the “South West Pacific” and “Queensland” community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 “Aus-1/Aus-2” hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Identification and Minisequencing-Based Discrimination of SHV β-Lactamases in Nosocomial Infection-Associated Klebsiella pneumoniae in Brisbane, Australia

ABSTRACT Extended-spectrum β-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV β-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the blaSHV-2a gene and seven strains carried the blaSHV-12 gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL blaSHV-11 gene and one strain carried the non-ESBL blaSHV-1 gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the blaSHV-2a or blaSHV12 gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed “first-nucleotide change,” involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying blaSHV-2a from strains carrying blaSHV-12. In addition, this method was used to demonstrate an association between the relative copy numbers of blaSHV genes in individual strains and the levels of antibiotic resistance.

blaSHV Genes in Klebsiella pneumoniae: Different Allele Distributions Are Associated with Different Promoters within Individual Isolates

ABSTRACT Extended-spectrum β-lactamases (ESBLs) emerge by point mutation from non-extended-spectrum precursors. The aims of this study were to reveal the basis for variations in resistance levels found in a collection of 21 Klebsiella pneumoniae clinical isolates from Brisbane, Australia. Previous studies have shown that 20 of these isolates possess blaSHV-11, blaSHV-2a, and/or blaSHV-12, and there is an association between the copy numbers of the ESBL-encoding genes and resistance levels. In this study, a real-time PCR method for interrogating the polymorphic sites at codons 238 and 240 was developed, and this confirmed the relationship between mutant gene copy numbers and resistance levels. The blaSHV promoter region was cloned from one of the ESBL-expressing isolates, and this showed that blaSHV genes exist downstream of two different promoters within this single isolate. These promoters have both been reported previously, and they differ by virtue of the presence or absence of an IS26 insertion. The blaSHV copy numbers in cis with the different promoters were measured, and the copy number of the IS26 promoter was correlated with resistance levels. Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with individual promoters in individual isolates but that mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL-positive isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL-positive phenotype.

First outbreak of PVL-positive nonmultiresistant MRSA in a neonatal ICU in Australia: comparison of MALDI-TOF and SNP-plus-binary gene typing.

The purpose of this brief report is to describe the first outbreak of a community-associated nonmultiresistant and PVL-positive MRSA strain (CC30) in a neonatal intensive care unit in Australia. The utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) for microbial typing is compared with single nucleotide polymorphism (SNP) plus binary gene analysis. The composite correlation index analysis of the MALDI-TOF-MS data demonstrated the similar inter-strain relatedness found with the SNP-plus-binary gene typing used to confirm the outbreak. The evolving spread of MRSA emphasizes the importance of surveillance, infection control vigilance and the ongoing investigation of rapid typing methods for MRSA.

Epidemiology of non-multiresistant methicillin-resistant Staphylococcus aureus infection in Queensland, Australia: associations with indigenous populations and Panton-Valentine leukocidin

The purpose of this study was to determine the extent of the spread of epidemic clones of non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) and the epidemiology of resultant infections throughout the state of Queensland. We collected a sample of clinical isolates of nmMRSA from laboratories serving public hospitals and clinics throughout the state. Three hundred isolates were typed and tested for the presence of Panton–Valentine leukocidin (PVL) genes and demographic and clinical data were collected from associated cases. Fifteen percent of S. aureus isolates were nmMRSA and 69% of these belonged to PVL-positive clones, predominantly ST93 and CC30. Low numbers of USA300- and USA400-like isolates were also present. Infections due to PVL-positive strains were much less frequently acquired in hospital (3.4%) than those due to PVL-negative nmMRSA (23.7%). Thirty-seven percent of cases were in indigenous people who make up only 3.6% of the general population. The proportion of cases with PVL-positive, but non-negative isolates decreased progressively with age, suggesting that immunity to PVL might be an important determinant of protection. nmMRSA strains are present throughout Queensland and cause infections in both community and healthcare settings.

Genotyping of Methicillin-Resistant Staphylococcus aureus by Assaying for the Presence of Variable Elements Associated with mecA

ABSTRACT The region surrounding mecA in methicillin-resistant Staphylococcus aureus (MRSA) is highly variable. We describe an approach for the rapid genotyping of MRSA by assaying for the presence or absence of variable or mobile elements previously shown to be associated with the mecA region.

A PCR method for the identification of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs

Aim: The aim of this study was to assess the discriminatory power and potential turn around time (TAT) of a PCR‐based method for the detection of methicillin‐resistant Staphylococcus aureus (MRSA) from screening swabs. Methods: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO‐direct). The PCR method involved pre‐incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre‐incubation in broth for 4 hours followed by culture on MSAO (MSAO‐broth). Results: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO‐direct, MSAO‐broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95%CI 51.9–83.3%), 98.6% (95%CI 97.1–100%), 84.6% (95%CI 76.2–100%) and 95.2% (95%CI 92.4–98.0%), respectively, and these results were almost identical to those obtained from MSAO‐direct. The agreement between MSAO‐direct and PCR was 61.5% (95%CI 42.8–80.2%) for positive results, 95.6% (95%CI 93.0–98.2%) for negative results and overall was 92.2% (95%CI 88.9–95.5%). Conclusions: (1) The discriminatory power of PCR and MSAO‐direct is similar but the level of agreement, especially for true positive results, is low. (2) The potential TAT for the PCR method provides a marked advantage over conventional methods. (3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real‐time PCR may lead to improvement in sensitivity and TAT.

Detection and characterisation of extended spectrum β-lactamases in Klebsiella pneumoniae causing nosocomial infection

Summary One hundred and ninety‐five multi‐resistant strains of Klebsiella pneumoniae were isolated at Princess Alexandra Hospital (PAH) between December 1991 and June 1995. All these organisms produced extended spectrum β‐lactamases (ESBLs) as detected by the double disc synergy test (DDST). Between June 1994 and June 1995, a second population of 67 multi‐resistant but DDST negative strains was isolated. Twenty multi‐resistant Klebsiella pneumoniae strains (16 DDST positive and four DDST negative) and one susceptible strain were selected for further study. These were tested for production of ESBLs by two double disc synergy methods and agar dilution minimum inhibitory concentrations (MICs) with and without clavulanic acid. Detected ESBLs were further characterised by isoelectric focusing. The confirmed DDST positive K. pneumoniae strains all produced ESBLs that focused at an isoelectric point (pl) of 7.6, suggesting the presence of SHV‐2, SHV‐2a, SHV‐6, SHV‐7 or SHV‐8 enzymes. The multi‐resistant DDST negative strains showed no clavulanic acid synergy and thus no evidence of the presence of ESBLs.Abbreviations: DDST, double disc synergy test; ESBL, extended spectrum β‐lactamase; PAH, Princess Alexandra Hospital.

Prevalence of the toxic shock gene (TST) in an Australian Staphylococcus aureus cohort and changes in strain prevalence and virulence genes, 1989–2003

Aim To determine the prevalence of toxic shock gene tst in an Australian Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) cohort collected over the last two decades. Method The identity of 300 S. aureus isolates was confirmed by real-time polymerase chain reaction (PCR) assays for mecA, nuc and 16S rRNA. Isolates were assayed for eight single nucleotide polymorphisms (SNPs) and for five binary genes (pvl, cna, sdrE, pUB110, pT181). SNP profiles are concordant with multilocus sequence typing (MLST) clonal complexes (CCs) and/or sequence types (STs). Two real-time PCR assays were developed for tst, based on primer sequences previously described. Results SNP profiles correlated with 21 CCs/STs. The 90 MRSA isolates correlated with three CCs: CC239, CC1 and CC22. We found 18 tst positive isolates, 3/91 (1989), 6/104 (1996) and 9/105 (2003). Of these, CC30 was predominant. Of 210 methicillin-sensitive S. aureus isolates, 26 were CC1, 24 CC5 and 23 CC78. Conclusions The proportion of tst positive isolates and binary genes including pvl was low, and has not increased significantly. Most tst positive isolates belong to CC30 which accords with overseas publications. In 1989 and 1996 CC239 MRSA (AUS-2/3) was the sole MRSA strain. MRSA decreased significantly in 2003 in spite of the appearance of CC1 MRSA (WA-1) and CC22 MRSA (EMRSA-15).