Stephen A. Neville

Utility of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry following Introduction for Routine Laboratory Bacterial Identification

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was evaluated prospectively in a diagnostic laboratory. Nine hundred twenty-seven organisms were tested in triplicate; 2,351/2,781 (85%) species and 2,681/2,781 (96%) genus identifications were correct. Known issues such as the misidentification of alpha-hemolytic streptococci as Streptococcus pneumoniae were easily corrected. Identifications cost AUD

Community-acquired, non-multiresistant oxacillin-resistant Staphylococcus aureus (NORSA) in South Western Sydney.

0.45 per isolate and were available in minutes. MALDI-TOF MS is rapid, accurate, and inexpensive.

Non-multiresistant methicillin-resistant Staphylococcus aureus bacteraemia in Sydney, Australia: emergence of EMRSA-15, Oceania, Queensland and Western Australian MRSA strains

Summary Community‐acquired oxacillin‐resistant Staphylococcus aureus (ORSA) infections are an emerging problem in the 1990s in Sydney, Australia. Laboratory data pertaining to all specimens that grew S. aureus between 1/1/1990 and 31/12/1999 were analysed. A total of 12,909 isolates of S. aureus were obtained. The proportions that were nonmultiresistant oxacillin‐resistant S. aureus (NORSA) increased from 0.09% in 1990 to 5.5% in 1999. Resistance of NORSA strains to erythromycin was 8.5%, ciprofloxacin 8.4%, tetracycline 13%, rifampicin 0.7%, and fusidic acid 5.3%. A chart review was performed for cases of NORSA infection which occurred 1/1/1998‐3/5/1998. Isolates from these cases underwent E‐test oxacillin MIC testing, mecA determinant PCR, phage typing and pulsed‐field gel electrophoresis. All nine of the patients with NORSA were Polynesians, and all had serious soft tissue infections. Bacteraemia was not seen. Only one patient received vancomycin yet all recovered. Isolates from all nine patients contained the mecA determinant. Oxacillin MICs were 1‐8 mg/l. Strain differentiation with phage typing and pulsed‐field gel electrophoresis showed isolates from eight patients were closely related and were similar to New Zealand WSPP 1 and WSPP 2 strains. Medical practitioners should take specimens for culture and sensitivity from lesions where infection with S. aureus is likely. Empirical treatment of staphylococcal infections in Polynesians needs to cover NORSA. Methods to detect oxacillin resistance need to be robust.

Evaluation of the MRSA-Screen Test in Detecting Oxacillin Resistance in Community and Hospital Isolates of Staphylococcus Aureus

Aims: To describe clinical features and molecular epidemiology of non‐multiresistant methicillin‐resistant Staphylococcus aureus (MRSA) bacteraemia. Methods: Patients with non‐multiresistant MRSA isolated from blood at South Western Area Pathology Service from 1 January 1999 to 31 December 2001 were enrolled. Pulsed field gel electrophoresis, phage typing, and (selected instances) multilocus sequence and staphylococcal cassette chromosome typing was performed. PCR was used to detect Panton‐Valentine leukocidin (PVL), toxic shock syndrome toxin‐1 (TSST‐1), and enterotoxin genes. Results: Sixteen patients were detected: eight with UK EMRSA‐15 (ST22‐MRSA‐IV), three with Oceania (South‐West Pacific/Western Samoan phage pattern) (ST30‐MRSA‐IV), two with WA MRSA‐5 (ST8‐MRSA‐IV), and one each with WA MRSA‐1 (ST1‐MRSA‐IV), Queensland strain (ST93‐MRSA‐IV), and WA MRSA‐15 (ST59‐MRSA‐IV). Prior hospital admissions occurred with six of the eight patients with UK EMRSA‐15, none of the three with Oceania, and three of the five with other strains. Thirteen of 16 patients had underlying disease. Three of the three patients with Oceania strain bacteraemia were Polynesians; 11 of 13 of the others were Caucasians. PVL genes were detected in four of 16 isolates (all Oceania and Queensland strains). entC was detected in two EMRSA‐15 strains; entA in one Oceania, two WA MRSA‐5 and the WA MRSA‐1 strain, with entA and entB in the WA MRSA‐15 strain. tst was not detected. Conclusions: Multiple epidemic strains cause non‐multiresistant MRSA bacteraemia. Most patients had risk factors. Oceania and Queensland strains possess the PVL gene. Abbreviations: ccr, cassette chromosome recombinase; EMRSA‐15, epidemic methicillin‐resistant Staphylococcus aureus‐15 EMRSA‐16, epidemic methicillin‐resistant Staphylococcus aureus‐16; MLST, multilocus sequence typing; MRSA, Methicillin‐resistant Staphylococcus aureus; PCR, polymerase chain reaction; PFGE, pulsed‐field gel electrophoresis; PVL, Panton‐Valentine leukocidin; RFLP, restriction fragment length polymorphism; SCCmec, staphylococcal cassette chromosome mec; STx‐MRSA‐IV, multilocus sequence type x‐MRSA‐SCCmec type‐IV; SWP, South‐West Pacific; TSST‐1, (staphylococcal) toxic shock syndrome toxin‐1; WA MRSA, Western Australian MRSA; WSPP, Western Samoan phage pattern.

Flavimonas Oryzihabitans Infection of a surgical wound

Summary The MRSA‐Screen Test (Denka Seiken Co., Japan), a latex agglutination test to detect penicillin‐binding protein 2a, was compared with PCR for the detection of oxacillin resistance in Staphylococcus aureus. A total of 77 oxacillin‐sensitive and 269 oxacillin‐resistant (ORSA) isolates were evaluated. Of the ORSA isolates, 186 were nonmultiresistant (NORSA), defined as being resistant to two or fewer antibiotics other than‐lactams. Eighty‐three were multiresistant ORSA (MORSA) strains. If PCR is considered the gold standard test, then the sensitivity, specificity, positive and negative predictive values of the MRSA‐Screen Test were 100, 99, 99 and 100%, respectively. The endpoint was hard to read with NORSA strains that took longer than 60 s to react. MORSA strains took a median 12 s (range 5‐60 s) to give a positive reaction with the MRSA‐Screen Test, whereas NORSA strains took a median 30 s (range 5‐180 s), a difference which was significantly different (P < 0.0001, two‐tailed Mann‐Whitney unpaired two sample test). NORSA strains had an MIC 50 of 128 mg/l and MIC 90 of 256 mg/l, whereas MORSA strains had an MIC 50 and MIC 90 of > 256 mg/l. The time that the MRSA‐Screen Test took to agglutinate with ORSA strains correlated weakly with the MIC (r 2 = 0.26). Detection of methicillin resistance cost AUD

An evaluation of the in vitro activity of piperacillin/tazobactam

9 per isolate with the MRSA‐Screen Test, compared with AUD

Rapid microbial identifications by MALDITOF : viva la revolution!

13 per isolate with mec A PCR. The MRSA‐Screen Test gave excellent sensitivity and specificity, and was quicker and cheaper than PCR. The full 3 min should be allowed to elapse before calling a test negative. Organisms giving indeterminate reactions should be tested for the mec A gene by PCR.

Non-multiresistant and multiresistant methicillin-resistant Staphylococcus aureus in community-acquired infections.

&NA; An unusual case of a post operative wound infection involving Flavimonas oryzihabitans is described. This organism is rarely isolated from human sources. It can cause infections in patients having continuous ambulatory peritoneal dialysis. Our patient developed a wound infection 2 months after femoro‐popliteal bypass grafting. The source of the organism was unknown.

Unusual Salmonella enterica serotype Typhimurium isolate producing CMY-7, SHV-9 and OXA-30 β-lactamases

&NA; Tazobactam is a new, irreversible inhibitor of bacterial betalactamases of staphylococci, plasmid‐mediated beta‐lactamases of the TEM and SHV types found in Escherichia coli and Klebsiella species and beta‐lactamases of anerobes such as Bacteroides species. Its combination with piperacillin, a broad spectrum ureido‐penicillin, would be expected to improve the activity of piperacillin against staphylococci, TEM and SHV betalactamase producing Gram negative bacteria and anerobes. Minimal inhibitory concentrations (MIC) of piperacillin/tazobactam were determined for 1952 individual patient isolates of Gram positive and negative bacteria causing significant infections and compared with MIC values for cefotaxime, ceftazidime, ciprofloxacin, imipenem, ticarcillin/clavulanic acid. MICs were determined by agar dilution (NCCLS 1990 and 1992). Piperacillin/tazobactam had excellent activity against methicillin susceptible staphylococci, Streptococcus pneumoniae, Haemophilus influenzae, enterococci and organisms of the Bacteroides fragilis group. It was also active against the majority of Enterobacteriaceae and Pseudomonas aeruginosa isolates tested. It was not active against extended spectrum beta‐lactamase (ESBL) producing Klebsiella species and some high level TEM and SHV beta‐lactamase producing E. coli and Klebsiella species. Activity against Gram negative organisms capable of producing chromosomally mediated beta‐lactamases was good, since in most organisms tested, the enzymes were not induced in sufficient quantities to cause antibiotic resistance. However some Enterobacter species were derepressed hyperproducing mutants; these isolates showed resistance to piperacillin/tazobactam since tazobactam does not inhibit these Class I beta lactamases. Activity was superior to ticarcillin/clavulanic acid for Gram negative rods. Imipenem was the most active agent against ESBL producing Klebsiella species. Piperacillin/tazobactam has a suitable spectrum of activity in vitro to suggest its use in monotherapy of mixed anerobic infections, mixed respiratory infections such as aspiration pneumonia and, in combination with an aminoglycoside, it would provide Gram positive as well as Gram negative cover of febrile episodes in immunosuppressed patients.

Clinical, pathologic and epidemiologic features of infection with Scedosporium prolificans: four cases and review

Although the application of mass spectrometry to bacterial identification was proposed as far back as 1975 by John Anhalt, it was not until 1987 when Professor Franz Hillenkamp and Dr Michael Karas first pioneered Matrix Assisted Laser Desorption/Ionisation Time-of-Flight mass spectrometry (MALDI-TOF MS) for truly rapid microbial identification in diagnostic microbiology laboratories. Conventional phenotypic methods of identifying isolates of bacteria, yeasts and filamentous fungi require hours to several days to complete, depending on the type of organism involved, and may be prone to error due to bias or inexperience. Even the more recent molecular innovations have their limitations that place them outside the scope and budget of many routine laboratories. However, MALDI-TOF MS technology has changed the way we think about microbial identifications and strain differentiation by providing results from plate to name in approximately five minutes for one isolate and around 90 minutes for 60 isolates at under