Joanne L. Mercer

Methicillin-resistant Staphylococcus aureus vancomycin susceptibility testing: methodology correlations, temporal trends and clonal patterns

OBJECTIVES To determine the correlation between various vancomycin MIC testing methodologies and explore the phenomenon of MIC creep. METHODS A total of 417 consecutive non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from Liverpool Hospital between 1997 and 2008 were retrieved. All isolates were classified using PFGE and underwent susceptibility testing for vancomycin using a standard Etest (AB bioMérieux, Solna, Sweden), Vitek2(®) (AST-P612; bioMérieux, Inc., Durham, NC, USA) and broth microdilution (BMD) performed as per the CLSI method. RESULTS Over the 12 years, 78% (n = 326) of the isolates were multiresistant MRSA (ST239-like by PFGE, where ST stands for sequence type). The correlation between MIC testing methods was moderate with Spearmans correlation coefficients of 0.50 for BMD versus Etest (P < 0.001), 0.33 for BMD versus Vitek2(®) (P < 0.001) and 0.42 for Etest versus Vitek2(®) (P < 0.001). In general, Etest results were 1 dilution higher while the Vitek2(®) results were 1 dilution lower than the BMD MIC result. MIC creep was dependent on the MIC testing method and the measurement used for analysis (geometric mean MIC versus modal MIC versus frequency analysis), with creep detected for Etest regression analysis only. In contrast, the proportion of isolates with a BMD MIC ≥2 mg/L decreased from 16% to 9% in the latter half of the study. Modal MIC was stable over the 12 years at 1 mg/L irrespective of MIC method used. CONCLUSIONS Correlation between vancomycin MIC methodologies remains suboptimal. Temporal MIC trends should be interpreted with caution as these are dependent on the testing methodology and the measurement used for analysis.

Performance of Various Testing Methodologies for Detection of Heteroresistant Vancomycin-Intermediate Staphylococcus aureus in Bloodstream Isolates

ABSTRACT The best screening method for detecting heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) remains unclear. Using population analysis profiling utilizing the area under the concentration-time curve (PAP-AUC) as the gold standard, we screened 458 consecutive methicillin-resistant S. aureus (MRSA) bloodstream isolates to determine the most accurate and cost-effective testing strategy to detect the presence of heteroresistance. All isolates were also tested using the macromethod Etest (MET) and glycopeptide resistance detection (GRD) Etest. The MIC was determined by several methods, including standard vancomycin Etest, vancomycin broth microdilution (BMD), and Vitek2 testing. Fifty-five (12%) hVISA and 4 (1%) VISA isolates were detected by PAP-AUC. Compared to PAP-AUC, the sensitivities and specificities of MET, GRD Etest, BMD (using a MIC cutoff of ≥2 mg/liter), and standard vancomycin Etest (using a MIC cutoff of ≥2 mg/liter) were 89 and 55%, 71 and 94%, 82 and 97%, and 71 and 94%, respectively. Combination testing increased the overall testing accuracy by reducing the number of false-positive results. Cost was determined predominately by the number of PAP-AUC runs required following a screening assay. The most cost-effective strategy was BMD (using a MIC cutoff of ≥2 μg/ml) as a standalone assay or in combination with PAP-AUC, provided that BMD testing was batched. GRD Etest remained an alternative, with 71% of hVISA isolates detected. Prevalence influenced both cost and test accuracy, with results remaining unchanged for hVISA prevalences of up to 25%. Implementation of any testing strategy would therefore be dependent on balancing cost with accuracy in a given population and clinical context.

Detection and serogroup determination of neisseria meningitidis in CSF by polymerase chain reaction (PCR)

Summary A PCR protocol for the detection and serogroup determination of Neisseria meningitidis in CSF from 85 cases of suspected meningitis was evaluated. Screening assays for both IS 1106 and the ctr A gene were used to detect meningococcal DNA, and a further two assays using the sia D gene were performed to determine the serogroup. PCR results were compared with results of bacteriological culture and discrepant results resolved by analysis of clinical data and further laboratory test results. The resolved sensitivity and specificity of the PCR screening assay were 89 and 100%, and those of bacteriological culture were 37 and 100%, respectively. The sia D B/C PCR assay was able to determine a serogroup in 85% of cases positive by the PCR screening assay compared with 50% of cases where a serogroup was determined by traditional methods. PCR is a useful tool for diagnosis of meningococcal meningitis when Gram stain and culture tests are negative, a situation that may arise when antibiotic treatment has commenced prior to lumbar puncture.

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

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.

Carriage of an ACME II Variant May Have Contributed to Methicillin-Resistant Staphylococcus aureus Sequence Type 239-Like Strain Replacement in Liverpool Hospital, Sydney, Australia

ABSTRACT Approximately 39% of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 239 (ST239)-like bloodstream isolates from Liverpool Hospital (obtained between 1997 and 2008) carry an arginine catabolic mobile element (ACME). Whole-genome sequencing revealed that an ACME II variant is located between orfX and SCCmec III, and based on pulsed-field gel electrophoresis patterns and temporal relationships of all ST239-like isolates (n = 360), ACME carriage may have contributed to subpulsotype strain replacement.

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

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.

Clinical features, epidemiology, antimicrobial resistance, and exotoxin genes (including that of Panton-Valentine leukocidin) of gentamicin-susceptible methicillin-resistant Staphylococcus aureus (GS-MRSA) isolated at a paediatric teaching hospital in New South Wales, Australia

Aims: To describe the epidemiological, clinical, and laboratory features of gentamicin‐susceptible methicillin‐resistant Staphylococcus aureus (GS‐MRSA) seen at a paediatric teaching hospital. Methods: Patients from whom GS‐MRSA was isolated between 1 January 2001 and 31 December 2002 were enrolled. Retrospective chart review was performed. Susceptibility testing was performed with the Vitek2 system; PCR confirmed methicillin resistance. Phage typing and pulsed field gel electrophoresis (PFGE) was performed (utilising MLST/SCCmec ‐defined control strains). PCR detection of tst, luk–PV, and entA‐entE was performed. Results: Eighty‐five per cent of all Staphylococcus aureus isolates during the study period were methicillin‐sensitive, and 15% were MRSA (9% GS‐MRSA, 6% gentamicin resistant‐MRSA). 100 GS‐MRSA infections in 98 children were identified: 59 cases of skin/soft tissue, four bone and joint, four surgical site infections, three pneumonia, eight other types, and 22 represented colonisation. Ninety‐nine isolates were non‐multidrug resistant, but 17 strains were resistant to erythromycin, 7 to tetracyclines, 12 to ciprofloxacin, 11 to fusidic acid, 1 each to rifampicin and mupirocin. 44 isolates were Oceania strain (ST30‐MRSA‐IV), 20 were Queensland strain (ST93‐MRSA‐IV), ten were UK EMRSA‐15 (ST22‐MRSA‐IV), eight were WA MRSA‐1 (ST1‐MRSA‐IV), two were WA MRSA‐5 (ST8‐MRSA‐IV), one was WA MRSA‐2 (ST78slv‐MRSA‐IV), one was WA MRSA‐15 (ST59‐MRSA‐IV), and the remainder were sporadics. Twenty patients were Pacific Islanders, of whom 12 had the Oceania strain; ten were Aboriginal, of whom eight had the Queensland strain. Sixty‐eight isolates possessed luk–PV, including all Queensland strains and 91% of Oceania strains. Enterotoxin genes were detected in 25% of the isolates, and tst was detected in four isolates. Conclusions: GS‐MRSA is a significant paediatric problem in New South Wales: two minority groups are over‐represented, multiple epidemic strains were detected, most community strains possess luk–PV, and many isolates are multidrug resistant.

Clostridium difficile Testing: Have We Got It Right?

We read with interest the recent article by Kaltsas et al. which retrospectively evaluated the impact of converting to a nucleic acid amplification test (NAAT)-based assay for Clostridium difficile detection ([1][1]). The authors described several possible consequences of such an approach as a

First Australian description of Eggerthella lenta bacteraemia identified by 16S rRNA gene sequencing

Sir, We wish to report the first Australian case of a patient with Eggerthella lenta infection, demonstrating the usefulness of 16S rRNA gene sequencing technology in a clinical diagnostic microbiology laboratory. A 68-year-old woman was admitted to hospital with a two-day history of malaise, anorexia, nausea and colicky abdominal pain. She had no fever or sweats, no change in bowel habit, no diarrhoea or vomiting and no melaena. She had a history of thyroidectomy, ectopic pregnancy and hypertension. On examination, the temperature was 37.48C, the blood pressure 98/58 mmHg and the pulse rate 86 beats per minute. She had generalised abdominal tenderness, but the rest of the examination was unremarkable. Initial radiological investigations showed no pneumoperitoneum, air-fluid levels or distended loops in the bowel. Ultrasound suggested the presence of free fluid in Morrison’s pouch. At laparotomy, purulent peritoneal fluid was seen and foulsmelling pus found around the uterus. The rest of the abdominal organs were macroscopically normal. There was evidence of previous appendicectomy. The patient underwent total abdominal hysterectomy, left salpingo-oophorectomy and irrigation of the peritoneum. The provisional diagnosis was a tubo-ovarian abscess. Intravenous ampicillin, gentamicin and metronidazole were commenced. Gram positive bacilli were isolated in pure growth from the blood cultures performed on admission and from a swab of the peritoneum taken at surgery. With initial improvement, the antibiotics were stopped. A week after admission, the patient deteriorated. CT scans demonstrated a pelvic collection with retroperitoneal extension. CT guided drainage of the collection yielded faecal fluid. She underwent resection of the sigmoid colon and Hartmann’s procedure. The antibiotics were recommenced. She developed a methicillin resistant Staphylococcus aureus (MRSA) bacteraemia. Her antibiotics were changed to vancomycin, timentin and metronidazole; however, she remained unwell. Three weeks after admission, she had further CT scans which showed a perisplenic collection, which yielded MRSA when drained. She gradually improved hereafter and was eventually discharged home 5 weeks after admission. Histological examination of the specimens obtained from the first surgical intervention confirmed a pyometrium with involvement of the fallopian tube and ovary. Examination of the specimens obtained from the sigmoid resection demonstrated diverticulosis of the sigmoid colon and localised peritonitis. Subculture of the Gram positive bacillus seen in the anaerobic blood culture bottle (BacT/ALERT 3D; bioMerieux, France) yielded small, low-convex, grey colonies on plates incubated anaerobically after 3 days. No growth was obtained on plates incubated aerobically. However, phenotypic identification did not proceed because the isolate was non-viable hereafter. The organism was subjected to 16S rRNA gene sequencing (MicroSeq500; Applied Biosystems, USA) and identified as Eggerthella lenta (98% identity over 478 bp to GenBank sequence AF292375). The isolate obtained from anaerobic cultures of the peritoneal swab was also non-viable on subculture. However, the primary plates had been discarded and thus were not available for further investigation. Eggerthella lenta is a member of the family Coriobacteriaceae. The differentiation and reclassification from other members of the genus Eubacterium was reported in 1999 on the basis of GþC content and 16S rRNA gene sequencing. Eubacterium lentum has a high GþC content (62 mol%) compared with the type species, Eubacterium limosum. Eggerthella lentum is found primarily in human faeces and is bile resistant, whereas Eubacterium exiguum is found predominantly in the mouth, in the context of periodontitis and periapical infections and is bile sensitive. 16S rRNA gene sequencing favoured the creation of a new genus, Eggerthella, to accommodate Eubacterium lentum, now renamed Eggerthella lenta gen. nov., comb. nov. The genus is named after Eggerth, the first person to isolate the organism. The cells are 0.2–0.4 6 0.2–2.0 mm, occur singly, in pairs and short chains and are Gram positive. Colonies are 0.5–2.0 mm, circular, raised to low-convex, translucent to semi-opaque, dull to shiny and smooth. The organism is an obligate anaerobe without spores or flagella. Glucose is fermented to produce acetate, lactate and succinate, phenotypically differentiating it from Propionibacterium (propionate), Lactobacillus (lactate), Actinomyces (succinate) and Bifidobacterium (acetate and lactate). Hydrogen is not produced. It is catalase negative, does not hydrolyse gelatin or aesculin but reduces nitrate. It is indole negative. In agar dilution studies performed according to National Committee of Clinical and Laboratory Standards (NCCLS) guidelines, around 40–50% of tested strains of Eggerthella lenta are inhibited by concentrations of 0.06 mg/mL or less, of amoxycillin, amoxycillin-clavulanate and clindamycin. Fewer strains still are inhibited at these concentrations of imipenem, metronidazole and moxifloxacin. The original strains of Eggerthella lenta were obtained from human faeces. Together with other anaerobes, it has also been isolated with a frequency of 50% from human appendices, whether non-inflamed, inflamed or gangrenous, as well as from the aortic walls from patients with abdominal aortic aneurysms. In both these conditions, it is unclear whether this association is pathogenic. The paucity of reported pathological associations is likely due to difficulty in cultivation and difficulty of differentiating these organisms from other non-sporulating anaerobic Gram positive bacilli. More recent studies, however, have begun to suggest both strong causal associations as well as importance in terms of frequencies. A group in Hong Kong found that Eggerthella species caused 18% of clinically significant bacteraemia due to anaerobic Gram positive bacilli. Half of these were due to E. lenta. There was a strong association with gastrointestinal disease with an Pathology (June 2008) 40(4), pp. 409–439

Ultrasound-enhanced latex immunoagglutination test (USELAT) for detection of capsular polysaccharide antigen of Neisseria meningitidis from CSF and plasma

Aim: An ultrasonic instrument, the Immunosonic, was used to evaluate ultrasound‐enhanced latex immunoagglutination testing (USELAT) for detection and serogroup determination of Neisseria meningitidis in clinical specimens. Methods: Eighty‐two CSF and EDTA blood specimens from patients with suspected meningococcal disease (MD) were tested by USELAT. Results were compared with routine laboratory tests for confirmation of MD and discrepant results were resolved by analysis of further laboratory and clinical data. Results: Using the Wellcogen Bacterial Antigen Kit, USELAT was positive in 20 (24%) specimens. The resolved sensitivity of USELAT was 49% compared with 67% for PCR. There were no discrepancies between serogroups indicated by USELAT and serogroups confirmed by PCR or culture grouping. Conclusions: Although USELAT could be performed in laboratories without facilities for PCR testing, a specific ultrasonic instrument is necessary and some experience is required in interpreting results. The lower resolved sensitivity makes USELAT unsuitable as a stand‐alone rapid test, and it added little value to standard laboratory culture with PCR testing.