Armine Sefton

High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells.

Staphylococcus aureus remains an important human pathogen responsible for a high burden of disease in healthcare and community settings. The emergence of multidrug-resistant strains is of increasing concern world-wide. The identification of S. aureus is currently based upon phenotypic and genotypic methods. Here, an alternative approach involving mass spectral analysis of surface-associated proteins of intact bacterial cells by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was investigated using 95 isolates obtained directly from a clinical laboratory at The Royal London Hospital and 39 isolates from the Staphylococcal Reference Unit, Health Protection Agency, London. Results obtained indicate that clinical isolates share many common mass ions with-type/reference strains which allowed their correct identification when searched against a comprehensive database that has been in the process of development for several years. The existing database contains more than 5000 profiles of various bacterial pathogens, but comprises mainly type or reference strains. The MicrobeLynx software successfully identified all isolates to the correct genus and all but four to the correct species. These were misidentified in the first instance due to contamination or low mass ion intensity but once the cultures were purified and re-analysed they were confirmed as S. aureus by both MALDI-TOF-MS and 16S rRNA sequence analysis. The high percentage of correct identifications coupled with the high speed and the minimal sample preparation required, indicate that MALDI-TOF-MS has the potential to perform high throughput identification of clinical isolates of S. aureus despite the inherent diversity of this species. The method is, however, only reproducible if variable parameters such as sample preparation, media, growth condition, etc. are standardised.

Multiple mutations modulate the function of dihydrofolate reductase in trimethoprim-resistant Streptococcus pneumoniae.

ABSTRACT Trimethoprim resistance in Streptococcus pneumoniaecan be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 μM) than was the DHFR from strain CP1015 (IC50, 0.09 μM). However,Km values indicated a lower affinity for the enzymes natural substrates (Km for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, Kmvalues for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates.

Septic shock, pneumonia, and soft tissue infection due to Myroides odoratimimus : report of a case and review of Myroides infections

The genus Myroides comprises aerobic, yellow-pigmented, non-motile, non-fermenting gram-negative rods formerly classified as Flavobacterium odoratum. Members of the genus are widely distributed in the environment, especially in water, and usually behave as low-grade opportunistic pathogens, having been found to cause urinary tract infection, endocarditis, ventriculitis, and cutaneous infections in severely immunocompromised patients. We report a case of soft tissue infection, septic shock, and pneumonia due to M. odoratimimus in an immunocompetent male. To our knowledge, this is the first description of life-threatening infection caused by this organism in an immunocompetent host. We have also reviewed the medical literature on the genus Myroides.

Macrolides and changes in the oral flora

Macrolides have been used in dental practice for many years, and may have a role in treating periodontal disease. Increased numbers of antibiotic-resistant oral streptococci have been reported after administration of both penicillins and macrolides. We confirm these findings for erythromycin, josamycin and azithromycin, and show that small numbers of macrolide-resistant streptococci are part of the normal oral flora at baseline. Resistant organisms fill the vacuum created by the removal of sensitive strains by antibiotic treatment. Following treatment with azithromycin, periodontal bacterial pathogens such as black pigmented anaerobes and spirochaetes decrease, whereas numbers of oral streptococci increase. These changes in the oral flora indicate a return to a healthier oral environment. In our studies, no clinical problems resulted from the transient increase in macrolide-resistant streptococci.

Epidemiology, antibiotic resistance trends and the cost of enteric fever in East London, 2005-2010.

INTRODUCTION Enteric fever seen in the UK has usually been acquired abroad. The cost to the NHS of treating enteric fever cases is not known. Data on the epidemiology of enteric fever, inpatient treatment costs and the public health management is needed to make decisions regarding the cost benefit considerations of introducing targeted prevention strategies. METHODS A retrospective study of laboratory confirmed enteric fever cases was conducted to estimate the cost of inpatient treatment and to determine antimicrobial resistance patterns at two hospitals in East London between January 2005 and the end of August 2010. RESULTS 138 cases of enteric fever were identified during the study period (90 S.ser.Typhi and 48 S. ser. Paratyphi). 92% had a recent history of foreign travel, 57% had travelled to visit friends and relatives (VFRs), 26% sought pre-travel health advice and 26% of patients had received typhoid vaccination. The inpatient treatment cost of 138 cases to the NHS was £272,747. The proportion of isolates with high level ciprofloxacin resistance (MICs>1 mg/L) has increased from 10% in 2006 to 30% in 2010. Our data also shows the emergence of isolates with high azithromycin MICs (>32 mg/L); 60% (six out of ten) isolates tested in July-August 2010. CONCLUSIONS There is a significant direct cost of treating enteric fever cases on the NHS. Cost reduction measures are confined due to the lack of effective oral antibiotics following the emergence of high level resistance to ciprofloxacin and azithromycin. Outpatient parenteral antibiotic therapy service and improved preventative public health measures aimed at VFR travellers in particular may be helpful in reducing costs.

Comparative in-vitro activity of penicillin alone and combined with gentamicin against clinical isolates of Streptococcus pneumoniae with decreased susceptibility to penicillin

The worldwide emergence of Streptococcus pneumoniae with decreased susceptibility to penicillin has led to the suggestion that drug combinations might be used. The aim of this study was to determine possible synergy using a combination of penicillin with sub-inhibitory doses of gentamicin against 26 clinical isolates of S. pneumoniae with decreased susceptibility to penicillin, using half-chequerboards and killing curves. Synergy was demonstrated for ten of the 26 isolates with the combination of penicillin with gentamicin at 1 mg/l and for 22 isolates with penicillin and gentamicin at 2 mg/l. Killing curves on three isolates showed synergy and confirmed the chequerboard results. Further synergy studies using penicillin or cefotaxime/ceftriaxone, plus low dose gentamicin against penicillin-resistant pneumococci are indicated.

Staphylococcus caprae meningitis following intraspinal device infection

A case is reported of Staphylococcus caprae meningitis due to infection of an intraspinal analgesia pump. The subclinical and pauci-symptomatic clinical course of the infection strongly suggested a chronic device contamination.

Enteric fever and its impact on returning travellers

Enteric fever, a systemic illness, is caused by Salmonella enterica serovar Typhi or S. enterica serovar Paratyphi A, B or C. The organism is transmitted to humans by the faecal oral route and is endemic in countries with poor sanitation and lacking clean drinking water. There are around 27 million individuals infected with S. Typhi worldwide annually. Enteric fever is a particular problem in travellers to endemic areas, especially those visiting friends and relatives. Currently, the two main vaccines recommended for travellers are the Vi polysaccharide vaccine and the oral Ty21a vaccine. These internationally licensed vaccines are safe and effective against S. Typhi. However, there is currently no commercially available vaccine against S. Paratyphi, which is increasingly reported as a cause of enteric fever. Vaccine uptake and taking appropriate precautions are poor in travellers visiting friends and relatives abroad; this problem requires addressing. Ciprofloxacin is no longer recommended for empirical treatment of infection because of increasing reports of resistance, especially from South Asia. Ceftriaxone and azithromycin are currently the most commonly used antimicrobials for empirical treatment of enteric fever but resistance to both these agents is emerging.

East London experience with enteric fever 2007-2012.

Purpose The clinical presentation and epidemiology for patients with enteric fever at two hospitals in East London during 2007–2012 is described with the aim to identify preventive opportunities and to reduce the cost of treatment. Methods A retrospective analysis of case notes from patients admitted with enteric fever during 2007 to 2012 with a microbiologically confirmed diagnosis was undertaken. Details on clinical presentation, travel history, demographic data, laboratory parameters, treatment, patient outcome and vaccination status were collected. Results Clinical case notes were available for 98/129 (76%) patients including 69 Salmonella enterica serovar Typhi (S. Typhi) and 29 Salmonella enterica serovar Paratyphi (S. Paratyphi). Thirty-four patients (35%) were discharged from emergency medicine without a diagnosis of enteric fever and then readmitted after positive blood cultures. Seventy-one of the 98 patients (72%) were UK residents who had travelled abroad, 23 (23%) were foreign visitors/new entrants to the UK and four (4%) had not travelled abroad. Enteric fever was not considered in the initial differential diagnosis for 48/98 (49%) cases. The median length of hospital stay was 7 days (range 0–57 days). The total cost of bed days for managing enteric fever was £454,000 in the two hospitals (mean £75,666/year). Median time to clinical resolution was five days (range 1–20). Seven of 98 (7%) patients were readmitted with relapsed or continued infection. Six of the 71 (8%) patients had received typhoid vaccination, 34 (48%) patients had not received vaccination, and for 31 cases (44%) vaccination status was unknown. Conclusions Further interventions regarding education and vaccination of travellers and recognition of the condition by emergency medicine clinicians in travellers to South Asia is required.

Trends in antibiotic susceptibility of enteric fever isolates in East London.

BACKGROUND The study sought evidence for changes in the proportions of antibiotic resistant strains among isolates of Salmonella enterica serovar Typhi (S. typhi) and Salmonella enterica serovar Paratyphi (S. paratyphi) between 2005 and 2012. METHODS Blood culture isolates of S. typhi and S. paratyphi from patients attending Newham and The Royal London Hospitals were included in the study. The organisms were cultured on selective media and identified by Maldi-ToF, API 20E and serology. Minimum inhibitory concentrations (MICs) of augmentin, chloramphenicol, co-trimoxazole, ceftriaxone, ciprofloxacin and azithromycin were determined by E tests for 194 isolates. RESULTS Median MICs of ciprofloxacin and ceftriaxone were stable at 0.5 mg/L and 0.125 mg/L, respectively. Chloramphenicol, azithromycin, co-trimoxazole and augmentin median MICs were 4 mg/L, 8 mg/L, 0.064 mg/L and 0.5 mg/L, respectively. MIC90 values were lower than the resistant breakpoint for ceftriaxone, azithromycin and augmentin, but were >256 mg/L for chloramphenicol, 32 mg/L for co-trimoxazole and 1 mg/L for ciprofloxacin. CONCLUSIONS Antibiotic resistance remained stable for enteric fever isolates between 2005 and 2012. The isolates remained susceptible to augmentin, ceftriaxone and azithromycin over this period, but the MIC90 was greater than the resistant breakpoint for chloramphenicol, cotrimoxazole and ciprofloxacin. The implications for clinical practice are that isolates of S. typhi and S. paratyphi from East London remain sensitive to ceftriaxone and azithromycin.