Time-Scaled Evolutionary Analysis of the Transmission and Antibiotic Resistance Dynamics of Staphylococcus aureus Clonal Complex 398.

Staphylococcus aureus clonal complex 398 (CC398) is associated with disease in humans and livestock, and its origins and transmission have generated considerable interest. We performed a time-scaled phylogenetic analysis of CC398, including sequenced isolates from the United Kingdom (Scotland), along with publicly available genomes. Using state-of-the-art methods for mapping traits onto phylogenies, we quantified transitions between host species to identify sink and source populations for CC398 and employed a novel approach to investigate the gain and loss of antibiotic resistance in CC398 over time. We identified distinct human- and livestock-associated CC398 clades and observed multiple transmissions of CC398 from livestock to humans and between countries, lending quantitative support to previous reports. Of note, we identified a subclade within the livestock-associated clade comprised of isolates from hospital environments and newborn babies, suggesting that livestock-associated CC398 is capable of onward transmission in hospitals. In addition, our analysis revealed significant differences in the dynamics of resistance to methicillin and tetracycline related to contrasting historical patterns of antibiotic usage between the livestock industry and human medicine. We also identified significant differences in patterns of gain and loss of different tetracycline resistance determinants, which we ascribe to epistatic interactions between the resistance genes and/or differences in the modes of inheritance of the resistance determinants.

Methicillin-resistant Staphylococcus aureus in Northeastern Scotland in 2003 to 2007: evolving strain distribution and resistance patterns.

This study explored strain distribution and resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) over a 5-year period in northeastern Scotland. We noted a shift in the relative rates of epidemic strains and an increase in community-associated strains. Use of oral antibiotics to eradicate throat carriage may have contributed to trimethoprim resistance, which was observed to increase 10-fold.

An unusual infection in an injecting drug user.

Injecting drug users are prone to atypical infections. We present a case of septic thrombophlebitis secondary to Fusobacterium gonidiaformans infection in a heroin user, which demonstrates the frequently unusual nature of pathogens and presentations in this group of patients.

Comparison of two multilocus variable-number tandem-repeat methods and pulsed-field gel electrophoresis for differentiating highly clonal methicillin-resistant Staphylococcus aureus isolates.

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson's index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.

Temporal analysis of invasive pneumococcal clones from Scotland illustrates fluctuations in diversity of serotype and genotype in the absence of pneumococcal conjugate vaccine.

In September 2006, the seven-valent pneumococcal conjugate vaccine (PCV7; Prevenar) was introduced into the childhood vaccination schedule in the United Kingdom. We monitored the population of invasive pneumococci in Scotland in the 5 years preceding the introduction of PCV7 by using serogrouping, multilocus sequence typing (MLST), and eBURST analysis. Here, we present a unique analysis of a complete national data set of invasive pneumococci over this time. We observed an increase in invasive pneumococcal disease (IPD) caused by serotypes 1, 4, and 6 and a decrease in serogroup 14-, 19-, and 23-associated disease. Analysis of sequence type (ST) data shows a significant increase in ST306, associated with serotype 1, and a decrease in ST124, associated with serotype 14. There have also been increases in the amounts of IPD caused by ST227 (serotype 1) and ST53 (serotype 8), although these increases were not found to reach significance (P = 0.08 and 0.06, respectively). In the course of the study period preceding the introduction of PCV7, we observed considerable and significant changes in serogroup and clonal distribution over time.

Investigation and control of a cluster of penicillin non-susceptible Streptococcus pneumoniae infections in a care home.

Two elderly residents of a care home were hospitalised with pneumonia over a period of one month. They had bacteraemia with penicillin non-susceptible Streptococcus pneumoniae (PNSP) and both died. All residents and staff of the care home were screened for PNSP using nasopharyngeal swabs, with one resident and one member of staff found to be asymptomatic carriers. Oral rifampicin was given to the carriers. All four strains were found to be serotype 14, and multilocus sequence typing (MLST) showed ST2652, not previously detected in Scotland. Review of care home residents showed that pneumococcal vaccination coverage was low (63%). This is similar to rates found in those aged > or =65 years in the general population and needs to be improved upon.

S-carboxymethylcysteine in the fluidification of sputum and treatment of chronic airway obstruction.

The clinical results and changes in sputum found in both a short-term inpatient trial and a subsequent long-term outpatient investigation (three-month double-blind controlled study) of 82 patients with chronic bronchitis treated with a new mucolytic agent, S-carboxymethylcysteine (Mucodyne), are reported. Fluidification of sputum with reduction in certain measurements of the viscosity of morning sputum aliquots, associated with improvement in the ability to cough up bronchial secretions, significant increase in sputum volume output, and improvement in ventilation (as estimated by the forced expiratory volume in one second), were observed in both trials as dose-related responses, with an increase in the ease of expectoration and a reduction in cough frequency and dyspnea. Therapy with S-carboxymethylcysteine was well tolerated, and there were no serious adverse effects, either immediate or delayed. We suggest that the effect of the drug in fluidifying sputum may be due to a mucoregulatory mechanism which reverses the sputum macromolecular disturbances seen in chronic bronchitis.

Laboratory confirmation of meningococcal disease in Scotland, 1993-9.

AIMS: To describe the laboratory confirmation of meningococcal disease, using culture and non-culture based techniques, between 1993 and 1999 as part of a national service in Scotland. METHODS: Samples from patients with suspected meningococcal disease in Scotland were analysed by culture and non-culture based techniques to gain a laboratory confirmation of disease. Data were analysed to establish the number of disease cases, the serogroups of the organisms involved, and the importance of the techniques used. RESULTS: Between 1993 and 1999, there was a total of 1749 notified cases of meningococcal disease in Scotland. Culture based methods provided a laboratory confirmation of 788 cases whereas non-culture techniques confirmed 461 cases. CONCLUSIONS: Non-culture techniques were a useful addition to culture based techniques in Scotland and improved the dataset required for public health management, disease surveillance, and vaccine policy.

Introduction of an automated service for the laboratory confirmation of meningococcal disease in Scotland.

The Scottish Meningococcus and Pneumococcus Reference Laboratory provides a national service for the laboratory confirmation of meningococcal and pneumococcal disease in Scotland. The main tests used for the laboratory confirmation of meningococcal disease are culture, the polymerase chain reaction (PCR), antibody testing, and more recently DNA sequencing. This paper describes the automation of PCR for the laboratory confirmation of meningococcal disease and the typing of meningococcal isolates using DNA sequencing. Both methods have been automated using a robotic liquid handler and automated DNA sequencer. These methods, along with standard culture phenotyping and antibody testing, provide Scotland with an excellent service for the confirmation of meningococcal disease.

Confirmation of meningococcal disease by urinary antigen testing.

The meningococcus is an important cause of morbidity and mortality and a rapid laboratory diagnosis is required through accurate, non-culture-based methods. Body fluids that are easily obtainable are preferred for this route of diagnosis and urine is the specimen of choice as it can be obtained non-invasively. Urine samples were tested from patients with suspected meningococcal disease and tested by latex agglutination and PCR. It was shown that urinary PCR is not useful for the laboratory confirmation of MD but latex agglutination testing may be useful in certain settings prior to confirmatory testing by a reference laboratory.

A bacteriological assessment of ampicillin with sulbactam as antibiotic prophylaxis in patients undergoing biliary tract operations. The West of Scotland Surgical Infection Study Group.

A prospective audit of 644 patients undergoing biliary tract operations has been conducted in ten district general hospitals. All patients received a single dose of ampicillin 2 g and sulbactam 1 g as antibiotic prophylaxis. Bacteria were cultured from the bile of 121 patients. In patients with sterile bile the incidence of postoperative infection was 2.5%, while in those with colonized bile it was 22% (P less than 0.0001). The 35 patients from whose bile bacteria of two or more species were isolated, had a higher incidence of wound infection (34%) than those whose bile yielded only one species of bacterium (17%; P less than 0.05). Seventeen of the 27 patients with colonized bile who developed postoperative infection were shown to be infected by the same organisms that had been isolated from their bile. The patients whose bile yielded organisms resistant to the prophylactic antibiotic combination did not have a significantly higher rate of infection than those from whose bile only sensitive organisms were obtained. A marked difference in sensitivity patterns between the participating hospitals was observed.

Automated non-culture-based sequence typing of meningococci from body fluids.

In recent years, the polymerase chain reaction has been used for the non-culture diagnosis of meningococcal disease, and sequence-based typing takes this further by providing the full characterisation normally only available by culture. In this study, porA gene sequencing was used to perform non-culture-based sequence typing of Neisseria meningitidis strains direct from body fluids. Non-culture porA gene sequencing provided the serosubtype of the infecting organism, and proved to be a useful method as N. meningitidis was not isolated from any of the patients in this study. In conclusion, porA gene sequencing is a very useful tool for the non-culture characterisation of meningococci and provides important information for public health management of cases and contacts.

Meningococcal disease due to serogroup Y in Scotland, 1992-1999.

Meningococcal disease is an important cause of morbidity and mortality. A retrospective analysis was performed of all cases of invasive group-Y disease that were laboratory-confirmed in Scotland between 1992 and 1999. A total of 1881 meningococcal isolates were characterised, 78 of which were serogroup Y. The incidence of non-invasive group-Y disease remained level between 1992 and 1999. Only 12 isolates were from invasive disease, comprising five strain types. Invasive group-Y disease was associated mostly with the young or old. Serogroup-Y meningococcal disease was uncommon and a rare cause of invasive disease in Scotland between 1992 and 1999; however, it is essential that microbiologists are aware of its potential for increasing in incidence due to the recent introduction of the MenC vaccine, and its increased incidence in the USA.

Meticillin-resistant Staphylococcus aureus (MRSA) update: New insights into bacterial adaptation and therapeutic targets.

Successful meticillin-resistant Staphylococcus aureus (MRSA) clones have evolved to adapt to healthcare, community and livestock environments. This review will bring together recent studies into clone adaptation and the importance of genes acquired during horizontal gene transfer to survival in specific environments. It will also discuss the role of global regulators controlling virulence gene expression and resistance to antibiotics, such as the agr and vraRS systems. Understanding these processes in successful clones could reveal novel targets for therapeutic agents, which are urgently required to reduce the infection burden and improve treatment options.

Legionella longbeachae serogroup 1 infections linked to potting compost.

Four cases of legionellosis caused by Legionella longbeachae serogroup (sg) 1 were identified in Scotland from 2008 to 2010. All case patients had exposure to commercially manufactured growing media or potting soils, commonly known as multipurpose compost (MPC), in greenhouse conditions, prior to disease onset. Two patients had been using the same brand of MPC but the clinical isolates were distinct genotypically by amplified fragment length polymorphism (AFLP) analysis. However, an indistinguishable AFLP profile was also found in an environmental isolate from the supply of MPC used by each patient. The third patient was diagnosed by immunofluorescent antibody serology only; however, the MPC to which this patient was exposed contained L. longbeachae sg 1 in large quantities (80 000 c.f.u. g(-1)). The fourth patient was L. longbeachae sg 1 culture-positive, but L. longbeachae was not identified from 10 samples of garden composting material. As compost is commonly used, but L. longbeachae infection seemingly rare, further work is required to ascertain (i) the prevalence and predictors of L. longbeachae in compost and (ii) the conditions which facilitate transmission and generate an aerosol of the bacteria. As most cases of legionellosis are diagnosed by urinary antigen that is Legionella pneumophila-specific and does not detect infection with L. longbeachae, patients in cases of community-acquired pneumonia with a history of compost exposure should have serum and respiratory samples sent to a specialist Legionella reference laboratory for analysis.

Enhanced isolation of Legionella species from composted material.

Legionella pneumophila and Legionella species were isolated from composted material when freshly prepared buffered charcoal yeast extract (BCYE) was supplemented with glycine (1.5 g/L), polymyxin B sulfate (40 000 IU/L), vancomycin hydrochloride (0.5 mg/L) and cycloheximide (40 mg/L) (GVPC medium) and Modified Wadowsky-Yee (MWY) (Oxoid, Cambridge, UK) plates were used for cultivation, but not with commercially sourced pre-poured GVPC and MWY plates (Oxoid). Legionella cincinnatiensis and pathogenic L. pneumophila serogroup (Sg) 1 Benidorm and France/Allentown were identified, as well as a non-typeable (NT) strain of L. pneumophila. As most laboratories no longer produce their own media, this may contribute to the lack of positive cultures from composted material. The antigenicity of the NT strain is discussed.

Decline and fall of epidemic meticillin-resistant Staphylococcus aureus-16.

Epidemic meticillin-resistant Staphylococcus aureus-16, which was widespread throughout the UK and the rest of the world, has declined markedly in recent years. The reasons for this are not clear.

A retrospective cohort study into acquisition of MRSA and associated risk factors after implementation of universal screening in Scottish hospitals.

OBJECTIVE: To estimate the proportion of patients who acquire methicillin-resistant Staphylococcus aureus (MRSA) while in hospital and to identify risk factors associated with acquisition of MRSA. DESIGN: Retrospective cohort study. PATIENTS: Adult patients discharged from 36 general specialty wards of 2 Scottish hospitals that had implemented universal screening for MRSA on admission. METHODS: Patients were screened for MRSA on discharge from hospital by using multisite body swabs that were tested by culture. Discharge screening results were linked to admission screening results. Genotyping was undertaken to identify newly acquired MRSA in MRSA-positive patients on admission. RESULTS: Of the 5,155 patients screened for MRSA on discharge, 2.9% (95% confidence interval [CI], 2.43-3.34) were found to be positive. In the subcohort screened on both admission and discharge (n = 2,724), 1.3% of all patients acquired MRSA while in hospital (incidence rate, 2.1/1,000 hospital bed-days in this cohort [95% CI, 1.5-2.9]), while 1.3% remained MRSA positive throughout hospital stay. Three risk factors for acquisition of MRSA were identified: age above 64 years, self-reported renal failure, and self-reported presence of open wounds. On a population level, the prevalence of MRSA colonization did not differ between admission and discharge. CONCLUSIONS: Cross-transmission of MRSA takes place in Scottish hospitals that have implemented universal screening for MRSA. This study reinforces the importance of infection prevention and control measures to prevent MRSA cross-transmission in hospitals; universal screening for MRSA on admission will in itself not be sufficient to reduce the number of MRSA colonizations and subsequent MRSA infections.