Meticillin-resistant Staphylococcus aureus in elderly residents of care homes: colonization rates and molecular epidemiology.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) is a significant cause of mortality and morbidity in healthcare and community settings; however, there is a paucity of large-scale, longitudinal studies monitoring the occurrence of MRSA in the care home setting. AIM: To determine the molecular epidemiology of MRSA colonizing elderly residents of care homes. METHODS: Residents in 65 care homes in Leeds, UK, were screened for MRSA nasal colonization in four consecutive years (2006-2009). Isolates were characterized using antibiotic susceptibility testing, detection of the Panton-Valentine leucocidin (PVL) locus, accessory gene regulator allotyping, characterization of the staphylococcal cassette chromosome mec element, spa-typing and pulsed-field gel electrophoresis. FINDINGS: MRSA was recovered from 888 nasal swabs of 2492 residents and prevalence was similar (19-22%) throughout the study. Resistance to ≥3 antibiotic classes was common (34%), but resistance to only ß-lactam agents was rare (3%); no PVL-positive isolates were identified. Most isolates were related to healthcare-associated epidemic-MRSA type 15 (EMRSA-15, ST22-IV); such isolates decreased in prevalence during the study (86-72%; P < 0.0001, χ(2)-test). The remainder belonged to five different multi-locus sequence type clonal complexes (CC). Most notably, CC59 strains increased in prevalence (10-25%; P < 0.0001, χ(2)-test) and were associated with high-level mupirocin resistance. CONCLUSIONS: The molecular epidemiology of MRSA in care homes is complex and dynamic. There was a high, consistent prevalence of MRSA nasal colonization, dominated by healthcare-associated strains. Vigilance is recommended; however, as high-level mupirocin resistance was associated with a single clonal group (CC59) that significantly increased in prevalence during the study.

Rapid updating of optical arbitrary waveforms via time-domain multiplexing.

We demonstrate high-fidelity optical arbitrary waveform generation with 5 GHz waveform switching via time-domain multiplexing. Compact, integrated waveform shapers based on silica arrayed-waveguide grating pairs with 10 GHz channel spacing are used to shape (line-by-line) two different waveforms from the output of a 10-mode x 10 GHz optical frequency comb generator. Characterization of the time multiplexer's complex transfer function (amplitude and phase) by frequency-resolved optical gating permits compensation of its impact on the switched waveforms and matching of the measured and target waveforms to better than G'=5%.

Use of multilocus sequence typing for the investigation of colonisation by Candida albicans in intensive care unit patients.

A prospective study was performed to determine the prevalence of candidal colonisation on the general intensive care unit at a large teaching hospital. Colonisation with Candida spp. was found to be common, occurring in 79% of patients on the unit. C. albicans was the commonest species, colonising 64% of patients, followed by C. glabrata (18%) and C. parapsilosis (14%). Most of the members of staff tested carried Candida spp. at some point, although carriage appeared to be transient. C. parapsilosis was the most commonly isolated species from staff hands, whereas C. albicans was the most commonly isolated species from the mouth. The molecular epidemiology of C. albicans was investigated using Ca3 typing and multilocus sequence typing (MLST). MLST proved to be a reproducible typing method and a useful tool for the investigation of the molecular epidemiology of C. albicans. The results of the molecular typing provided evidence for the presence of an endemic strain on the unit, which was isolated repeatedly from patients and staff. This finding suggests horizontal transmission of C. albicans on the unit though it may also reflect the relative frequency of C. albicans strain types colonising patients on admission. This study has important implications for the epidemiology of systemic candidal infections.

32 Phase X 32 amplitude optical arbitrary waveform generation.

We describe the precise shaping and mode-resolved amplitude and phase characterization of optical arbitrary waveforms by using a 20 GHz optical frequency comb and integrated 64 x 20 GHz channel arrayed waveguide grating pair. Complex waveforms with large variations in phase and amplitude between adjacent modes were generated and characterized.

High-fidelity line-by-line optical waveform generation and complete characterization using FROG.

A stable optical frequency comb with 20-GHz spacing is shaped by a compact integrated silica arrayed waveguide grating (AWG) pair to produce optical waveforms with unprecedented fidelity. Complete characterization of both the intensity and phase of the crafted optical fields is accomplished with cross-correlation frequency resolved optical gating (XFROG) which has been optimized for periodic waveforms with resolvable modes. A new method is proposed to quantify, in a single number, the quality of the match in both the amplitude and phase between the measured optical waveform and the target waveform.

SHV-12, SHV-5, SHV-2a and VEB-1 extended-spectrum beta-lactamases in Gram-negative bacteria isolated in a university hospital in Thailand.

Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P. aeruginosa had bla(SHV-12). Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread. At least six different bla(VEB-like-)containing integrons were found among the 18 isolates. This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P. aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P. putida. These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli.

A comparison of culture and PCR to determine the prevalence of ampicillin-resistant bacteria in the faecal flora of general practice patients.

Of 95 faecal specimens containing bacterial DNA amplified by PCR, 24% contained cultivable bacteria that were resistant to high-level ampicillin. When these samples were examined by PCR using primers to amplify the bla(TEM) gene, the number of positive samples identified increased significantly to 49 (52%). These results indicate that ampicillin resistance is common in the study population. Furthermore, the bla(TEM) gene is more common than indicated by the recovery of resistant bacteria in culture. This points to potential anomalies in the assessment of the prevalence of resistance when relying on recovery of resistant bacteria by culture.

Discrimination of SHV beta-lactamase genes by restriction site insertion-PCR.

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3' end to modulate a restriction site. We have developed this technique to identify described mutations of the bla(SHV) genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV beta-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly-->Ala) (BsrI), and 240 (NruI) of bla(SHV) genes. All amplimers of the bla(SHV) genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(SHV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV beta-lactamases and may be extended to the characterisation of other resistance determinants.

Survival of free DNA encoding antibiotic resistance from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and silage effluent.

To assess the likelihood that the bla gene present in a transgenic maize line may transfer from plant material to the microflora associated with animal feeds, we have examined the survival of free DNA in maize silage effluent, ovine rumen fluid and ovine saliva. Plasmid DNA that had previously been exposed to freshly sampled ovine saliva was capable of transforming competent Escherichia coli cells to ampicillin resistance even after 24 h, implying that DNA released from the diet could provide a source of transforming DNA in the oral cavity of sheep. Although target DNA sequences could be amplified by polymerase chain reaction from plasmid DNA after a 30-min incubation in silage effluent and rumen contents, only short term biological activity, lasting less than 1 min, was observed in these environments, as shown by transformation to antibiotic resistance. These experiments were performed under in vitro conditions; therefore further studies are needed to elucidate the biological significance of free DNA in the rumen and oral cavities of sheep and in silage effluent.

Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP).

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.

Effects of optical layer impairments on 2.5 Gb/s optical CDMA transmission.

We conducted a computer simulation study to assess the effects of optical layer impairments on optical CDMA (O-CDMA) transmission of 8 asynchronous users at 2.5 Gb/s each user over a 214-km link. It was found that with group velocity dispersion compensation, two other residual effects, namely, the nonzero chromatic dispersion slope of the single mode fiber (which causes skew) and the non-uniform EDFA gain (which causes interference power level to exceed signal power level of some codes) degrade the signal to multi-access interference (MAI) ratio. In contrast, four wave mixing and modulation due to the Kerr and Raman contributions to the fiber nonlinear refractive index are less important. Current wavelength-division multiplexing (WDM) technologies, including dispersion management, EDFA gain flattening, and 3 rd order dispersion compensation, are sufficient to overcome the impairments to the O-CDMA transmission system that we considered.

Evolution and spread of SHV extended-spectrum beta-lactamases in gram-negative bacteria.

Resistance to beta-lactam antibiotics has been a problem for as long as these drugs have been used in clinical practice. In clinically significant bacteria the most important mechanism of resistance is the production of one or more beta-lactamases, enzymes that hydrolyse the beta-lactam bond characteristic of this family of antibiotics. Prominent among the beta-lactamases produced by the Enterobacteriaceae is the SHV family. The first reported SHV beta-lactamase had a narrow spectrum of activity. By the accumulation of point mutations at sites that affect the active site of the enzyme, a family of derivatives of SHV-1 has evolved. Derivatives of SHV-1 either have an extended spectrum of activity, capable of inactivating third-generation cephalosporins, or are resistant to beta-lactamase inhibitors. This review describes the evolution and spread of the SHV family of beta-lactamases, introducing the structure-function analysis made possible by DNA sequence analysis. It also reviews the methods used to characterize members of this family of beta-lactamases, indicating some of the difficulties involved.

Online commentary in acute medical visits: a method of shaping patient expectations.

This paper conceptualizes a type of physician communication, termed 'online commentary'. Online commentary is talk that describes what the physician is seeing, feeling or hearing during physical examination of the patient. Some dimensions of online commentary are described, and its functions in routine and acute medical consultations are distinguished. Using a case study method, the paper focuses on the role of online commentary in pre-empting patient resistance to upcoming 'no problem' diagnostic evaluations which could delegitimize patients' decisions to seek medical assistance, or deprive them of anticipated medical benefits. It is hypothesized that this role for online commentary may be associated with successful physician resistance to implicit or explicit patient demands for inappropriate antibiotic medication.

Comparison of ARDRA and recA-RFLP analysis for genomic species identification of Acinetobacter spp.

The genus Acinetobacter is subdivided into genospecies on the basis of DNA relatedness of strains. Phenotypic tests are insufficient to identify the genospecies to which an isolate belongs. The effectiveness of two previously described PCR-based methods for genospeciating Acinetobacter spp. was compared using a group of 32 well-characterised strains representing six genospecies. Amplified ribosomal DNA restriction analysis (ARDRA) correctly identified all 32 strains. Using restriction fragment length polymorphism (RFLP) of recA PCR amplimers, only six of the 32 strains were correctly identified. Heterogeneity in the recA gene sequence was demonstrated within five of the genospecies. ARDRA proved to be a reliable method whereas analysis of recA RFLP profiles did not enable the genospecies of most of the isolates of Acinetobacter spp. to be determined.

Exceptional desiccation tolerance of Acinetobacter radioresistens.

The taxonomy of the genus Acinetobacter, which includes several important nosocomial pathogens, has been confused due to a lack of discriminatory phenotypic characteristics for identification. Molecular methods such as amplified ribosomal DNA restriction analysis (ARDRA) now enable the accurate identification of species. Ten clinical isolates of Acinetobacter radioresistens had genospecies confirmed by ARDRA but the APJ 20NE system, commonly used in clinical microbiology laboratories, mis-identified them as Acinetobacter lwoffii. Desiccation resistance of Acinetobacter spp. is an important attribute for their survival in the clinical environment. We investigated the ability of A. radioresistens to survive desiccation using an established glass surface model and compared the results to A. lwoffii and Acinetobacter baumannii. The 10 strains of A. radioresistens were extremely resistant to desiccation and survived for an average of 157 days at 31% relative humidity (RH). In contrast, two strains of A. lwoffii and three strains of A. baumannii survived for an average of three and 20 days respectively, at 31% RH, which was used as an approximation to climatic conditions in UK hospitals. A. radioresistens is thus well adapted for survival in the hospital environment and carriage on human skin and yet it is reported less frequently than A. lwoffii amongst clinical isolates. Cases of A. radioresistens infection may be under-reported due to mis-identification as A. lwoffii and further studies that use molecular identification methods are required to elucidate the role of A. radioresistens in human disease.

Survival of Acinetobacter baumannii on dry surfaces: comparison of outbreak and sporadic isolates.

Acinetobacter spp. are important nosocomial pathogens reported with increasing frequency in outbreaks of cross-infection during the past 2 decades. The majority of such outbreaks are caused by Acinetobacter baumannii. To investigate whether desiccation tolerance may be involved in the ability of certain strains of A. baumannii to cause hospital outbreaks, a blind study was carried out with 39 epidemiologically well-characterized clinical isolates of A. baumannii for which survival times were determined under simulated hospital conditions. The survival times on glass coverslips of 22 strains isolated from eight well-defined hospital outbreaks in a German metropolitan area were compared with the survival times of 17 sporadic strains not involved in outbreaks but rather isolated from inpatients in the same geographic area. All sporadic isolates have been shown by pulsed-field gel electrophoresis to represent different strain types. There was no statistically significant difference between the survival times of sporadic strains of A. baumannii and outbreak strains (27.2 versus 26.5 days, respectively; P < or = 0.44) by the Wilcoxon-Mann-Whitney test. All investigated A. baumannii strains, irrespective of their areas of endemicity or epidemic occurrence, have the ability to survive for a long time on dry surfaces. Antimicrobial susceptibility testing showed that A. baumannii outbreak strains were significantly more resistant to various broad-spectrum antimicrobial agents than sporadic strains. Both desiccation tolerance and multidrug resistance may contribute to their maintenance in the hospital setting and may explain in part their propensity to cause prolonged outbreaks of nosocomial infection.