Assessment of multiple coagulase-negative staphylococci isolated in blood cultures using pulsed-field gel electrophoresis

One of the criteria used to determine the clinical importance of coagulase-negative staphylococci (CMS) is the isolation of the bacteria from sequential blood cultures. Sequential isolates of CNS obtained from five immunocompromised patients over three months were genetically characterized by pulsed-field gel electrophoresis (PFGE). This typing method was compared to two first-line typing methods: determination of the species and of antibiotic susceptibility. In four patients the initial clinical evaluation changed because of the PFGE results; several episodes of bacteremia would have been wrongly assessed if only the biotype and the antibiotype had been determined. Pulsed-field gel electrophoresis should therefore be used for CNS strains from immunocompromised patients or those suffering from chronic diseases with non-concordant biotype and antibiotype.     

Catheter-related bloodstream infection caused by Enterococcus spp.

The role of Enterococcus spp. as a cause of catheter-related bloodstream infections (CR-BSI) is almost unexplored. We assessed the incidence and clinical characteristics of enterococcal CR-BSI (ECR-BSI) over an 8-year period in our hospital. We performed a retrospective study (January 2003 to December 2010) in a large teaching institution. We recorded the incidence, and the microbiological and clinical data from patients with ECR-BSI. The incidence per 10 000 admissions for enterococcal BSI and ECR-BSI was 25 and 1.7, respectively. ECR-BSI was the fourth leading cause of CR-BSI in our institution (6%). A total of 75 episodes of ECR-BSI were detected in 73 patients (6% of all enterococcal BSI). The incidence of ECR-BSI increased by 17% annually (95% CI 19.0–21.0%) during the study period. Nineteen percent of ECR-BSI episodes were polymicrobial. Overall mortality was 33%. ECR-BSI is an emerging and increasingly common entity with a high mortality. This finding should be taken into account when selecting empirical treatment for presumptive CR-BSI.     

Rapid diagnosis of catheter-related bloodstream infection using bioluminescence assay

Rapid diagnosis of the central venous catheter-related bloodstream infection (CR-BSI) is especially crucial in critical care settings. In this study, we applied bioluminescence assay method (BA method) to measure the levels of ATP, as a representative of bacterial count of the catheter tip. The efficacy of BA method was evaluated in comparison with Brun-Buisson method (BB method), a commonly used, semi-quantitative microbiological culture technique. Significant differences were detected in the ATP levels by BA method, 177 [935] vs. 1337 [5198] RLU(median [interquartile range: IQR]) between BB-negative and BB-positive group (p = 0.012). The sensitivity/specificity for the detection of BB-positive results was 88.9%/68.8% and 100.0%/65.7%, if we set the cutoff value of BA method as 500 and 300 RLU, respectively. BA method, a less time-consuming technique, could become a useful option for the rapid screening for CR-BSI.     

Evolution and aetiological shift of catheter-related bloodstream infection in a whole institution: the microbiology department may act as a watchtower

The incidence of central-line-associated bloodstream infection (CLA-BSI) is reported per 1000 days of catheter exposure, mainly in the intensive care unit (ICU), because recording exposure throughout an institution is not always feasible. Confirmation of catheter-related bloodstream infection (CR-BSI) requires specific laboratory testing that identifies the catheter as the source of infection. This information is available in microbiology laboratories and can be assessed using a denominator of 1000 admissions. We evaluated recent trends in the incidence and aetiology of CR-BSI and compared adult ICUs with the remaining areas of the hospital in a retrospective cohort analysis of all confirmed CR-BSIs. During the 8-year study period, we recorded 1208 episodes (8.2% of BSIs) of CR-BSI. After adjusting for the blood cultures drawn, a significant reduction in incidence was observed in adult ICUs (47%), where care bundles had been applied. The reduction was similar irrespective of whether CLA-BSI or CR-BSI was assessed. We recorded a significant reduction in the incidence of Staphylococcus aureus CR-BSI, and a significant increase in the incidence of CR-BSI caused by Enterococcus sp., Gram-negative microorganisms and fungi. The microbiology department may complement CLA-BSI/1000 catheter-days by providing CR-BSI when days of exposure are not available, because both figures are parallel. We demonstrated a significant reduction in the incidence of CR-BSI in recent years in the population admitted to adult ICUs but not in the remaining areas of the hospital. A shift in the aetiological spectrum of CR-BSI may be occurring.     

Reduction in catheter-related bloodstream infections in critically ill patients through a multiple system intervention

In this study, we aimed to determine the utility of a multiple system intervention to reduce catheter-related bloodstream infections (CR-BSI) in our intensive care unit (ICU). A prospective cohort study was undertaken in the medical and surgical ICU at a university hospital. We applied five measures: educational sessions about inserting and maintaining central venous catheters, skin cleaning with chlorhexidine, a checklist during catheter insertion, subclavian vein insertion and avoiding femoral insertion whenever possible, and removing unnecessary catheters. We determined the rate of CR-BSI per 1,000 catheter-days during the intervention (March to December 2007) and compared it with the rate during the same period in 2006 in which we applied only conventional preventive measures. CR-BSI was defined as the recovery of the same organism (same species, same antibiotic susceptibility profile) from catheter tip and blood cultures. We registered 4,289 patient-days and 3,572 catheter-days in the control period and 4,174 patient-days and 3,296 catheter-days in the intervention period. No significant differences in the number of patients with central venous catheters during the two periods were observed: catheters were used in 81.5% of patients during the control period and in 80.6% of patients during the intervention period. During the control period, 24 CR-BSI were diagnosed (6.7/1,000 catheter-days); during the intervention period, 8 CR-BSI were diagnosed (2.4/1,000 catheter-days) (relative risk 0.36; 95% confidence interval [CI] 0.16 to 0.80; p = 0.015). Nurses interrupted the procedure to correct at least one aspect when completing the checklist in 17.7% of insertions. In conclusion, a multiple system intervention applying evidence-based measures reduced the incidence of CR-BSI in our ICU.     

Central venous catheter-related blood stream infections in patients receiving intravenous iloprost for pulmonary hypertension

Catheter-related blood stream infection (CR-BSI) in patients with pulmonary hypertension (PH) receiving intravenous iloprost via an indwelling central line has previously not been fully described. Recent studies have suggested a link between the pH of prostanoid infusions and the rate and nature of CR-BSI. We have investigated CR-BSI in patients receiving intravenous iloprost at our unit. Databases and hospital records were interrogated for all patients receiving intravenous iloprost between September 2007 and June 2012. Fifty-nine patients received intravenous iloprost via an indwelling central catheter with a total of 23,072 treatment days. There were 15 episodes of CR-BSI, identified using a systematic screening protocol, involving 11 patients giving an overall CR-BSI rate of 0.65/1,000 treatment days. CR-BSI rate for Gram-positive organisms was 0.26/1,000 treatment-days and for Gram-negative organisms was 0.39/1,000 treatment-days. The pH of iloprost in typical dosing regimens was comparable to the pH used in standard-diluent treprostinil and dissimilar to alkaline epoprostenol infusions. The proportion of Gram-negative CR-BSI was similar to that reported for standard-diluent treprostinil. CRP was normal on admission in 33 % of cases of confirmed CR-BSI and remained normal in 13 % of cases. CR-BSI rates with intravenous iloprost are comparable to those observed for other prostanoids. The high proportion of Gram-negative organisms observed and the neutral pH of iloprost infusions support the previously hypothesised link between pH and antimicrobial activity. Although usually elevated during a CR-BSI, CRP may be normal in early infection and a normal result cannot completely exclude infection.     

Typing of Staphylococcus epidermidis clinical strains by a selected panel of Brazilian killer yeasts

Discrimination of multi-resistant microorganisms in small clinical microbiology laboratories is frequently based on the biologic profile (biotype) of phenotypic markers, such as antimicrobial susceptibility patterns (antibiograms) and serologic or enzymatic typing, but few use indicative microorganisms. The purpose of this study was to evaluate the power of a panel of selected killer yeasts for differentiating and discriminating clinical isolates of Staphylococcus epidermidis from two care hospitals and clinical microbiology laboratories from the South of Brazil. The short panel of eleven killer yeasts was capable of discriminating 100% of the sensitive strains of S. epidermidis using quantitative data matrix (QDM) and differentiating them from strains of coagulase-positive Staphylococcus. Therefore, this phenotypic methodology proved to be valid as a discriminatory tool when applied to these clinical bacteria strains, besides being simple and feasible for routine use even in microbiological laboratories with minimal resources.     

Characterization of Extended-Spectrum β-Lactamase-Producing Salmonella typhimurium by Phenotypic and Genotypic Typing Methods

During 1994, 10 isolates of extended-spectrum beta-lactamase-producing Salmonella typhimurium were recovered from children transferred to our hospital from two different centers. Two additional isolates were recovered from two nurses from one of these centers. The aim of this study was to determine if there is any relationship between these isolates. The characterization was done by phenotypic and genotypic methods: biotyping, phage typing, antibiotic susceptibility pattern determination, plasmid analysis, ribotyping (by the four endonucleases EcoRI, SmaI, BglII, and PvuII), pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with XbaI, and randomly amplified polymorphic DNA (RAPD) pattern determination (with the three primers 217 d2, B1, and A3). The same biotype, the same serotype, and an identical antibiotype were found. All isolates were resistant to oxyimino-beta-lactams, gentamicin, tobramycin, and sulfamethoxazole-trimethoprim. All isolates showed an indistinguishable pattern by ribotyping and very similar patterns by PFGE and RAPD. The overall results indicated the spread of a closely related strain of S. typhimurium in children and nurses.     

16S ribosomal DNA sequence analysis distinguishes biotypes of Streptococcus bovis: Streptococcus bovis Biotype II/2 is a separate genospecies and the predominant clinical isolate in adult males

We characterized 22 human clinical strains of Streptococcus bovis by genotypic (16S rRNA gene sequence analysis [MicroSeq]; Applied Biosystems, Foster City, Calif.) and phenotypic (API 20 Strep and Rapid ID32 Strep systems (bioMerieux Vitek, Hazelton, Mo.) methods. The strains, isolated from blood, cerebrospinal fluid (CSF), and urine, formed two distinct 16S ribosomal DNA sequence clusters. Three strains which were associated with endocarditis urinary tract infection (UTI), and sepsis clustered with the S. bovis type strain ATCC 33317 (cluster 1); other closely related type strains were S. equinus and S. infantarius. Nineteen strains clustered at a distance of about 2.5% dissimilarity to the S. bovis type strain (cluster 2) and were associated with central nervous system (CNS) disease in addition to endocarditis, UTI, and sepsis. All strains were distinct from S. gallolyticus. Within cluster 2, a single strain grouped with ATCC strain 43143 (cluster 2a) and may be phenotypically distinct. All the other strains formed a second subgroup (cluster 2b) that was biochemically similar to S. bovis biotype II/2 (mannitol negative and beta galactosidase, alpha galactosidase, beta glucuronidase, and trehalose positive). The API 20 Strep system identified isolates of cluster 2b as S. bovis biotype II/2, those of cluster 1 as S. bovis biotype II/1, and that of cluster 2a as S. bovis biotype I. There was an excellent correlation of biotype and genotype: S. bovis biotype II/2 isolates form a separate genospecies distinct from the S. bovis, S. gallolyticus, and S. infantarius type strains and are the most common isolates in adult males.     

Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases

The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.     

Strong slime production is a marker of clinical significance in <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> isolated from intravascular catheters

Biofilm production was assessed in 52 Staphylococcus epidermidis isolates from the catheters of 52 patients with catheter-related bloodstream infections (CR-BSI) and compared with 14 isolates from the skin of healthy volunteers by spectrophotometry. The isolates were classified as non- (G1), weak- (G2) or strong- (G3) slime producers based on optical density, and as producers and non-producers based on the results of the Congo red agar test. Differences (p = 0.012) in the proportion of G1, G2 and G3 among the isolates were found between catheter and healthy skin strains: there was a higher percentage of G1 types among the healthy skin strains (35.7 vs. 11.5%; p = 0.046) and a higher percentage of G3 types among the catheter isolates (44.2 vs. 0%; p = 0.001). No significant differences were found with the Congo red agar test. G3 is a phenotypic marker for CR-BSI.     

Phenotypic and genotypic characterisation of blood isolates of coagulase-negative staphylococci in the newborn

Coagulase-negative staphylococci (CNS) are the leading cause of late-onset sepsis in newborns (>72 h of age). Our aim was to determine whether phenotypic and/or genotypic differences existed between blood isolates of CNS regarded as inducers of sepsis or as contaminants. Ninety-seven bloodisolates of CNS recovered from newborns at the neonatal intensive care unit, Orebro, Sweden in 1983-1997 were analysed. Twenty-nine of them (30%) were classified as sepsis isolates and 68 (70%) as contaminants. The most prevalent species was Staphylococcus epidermidis (n=59). Staphylococcus haemolyticus (n=16) was most often isolated from newborns with the lowest gestational age and birth weight. Biochemical typing using the Phene Plate system (PhP) and genotyping using pulsed-field gel electrophoresis (PFGE) showed that the S. epidermidis isolates regarded as inducers of sepsis (n=16) were more homogeneous than isolates considered contaminants (n=37). One main genotypic group, representing seven (44%) isolates, was identified among the sepsis isolates. Phenotypically the S. epidermidis sepsis isolates comprised three major clusters. In contrast, among the S. epidermidis contaminants, eight genotypic groups and two phenotypic clusters were identified. The dominating genotypic group among the sepsis isolates of S. epidermidis may represent strains with higher invasive capacity.     

Revisited distribution of nonfermenting Gram-negative bacilli clinical isolates

Nonfermenting Gram-negative bacilli (NF-GNB) are ubiquitous environmental opportunistic bacteria frequently misidentified by conventional phenotypic methods. The aim of this study was to determine the distribution of NF-GNB species by 16?S rRNA gene sequencing (used as reference method) and to compare performances of biochemical tests and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). From nine French hospitals, 188 NF-GNB isolates (except P. aeruginosa and A. baumannii) were prospectively collected from 187 clinical samples between December 2008 and May 2009. By using the genotypic approach, 173 (92%) and 188 (100%) isolates were identified to the species and genus level, respectively. They covered 35 species and 20 genera, with a predominance of Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Pseudomonas putida group bacteria. Of the 173 species-level identified strains, concordant identification to the species-level was obtained for 75.1%, 83% and 88.9% of isolates with API 20 NE strip, the VITEK-2 (ID-GN card) system and MALDI-TOF-MS, respectively. By excluding S. maltophilia isolates accurately identified by the three methods, genus-level identification was much higher for MALDI-TOF-MS (92.9%), compared with API 20 NE and VITEK-2 (76.2% and 80.8%, respectively). In conclusion, MALDI-TOF-MS represents a rapid, inexpensive, and accurate tool for routine identification of NF-GNB in human clinical samples.     

Clinical features and therapeutic interventions in 17 cases of Bacillus bacteremia in an immunosuppressed patient population.

We retrospectively examined episodes of Bacillus bacteremia at a hospital with a large proportion of immunosuppressed patients. Seventeen episodes in 9.5 years met our case definition: two of two bottles of one blood culture or one of two bottles of two or more separately obtained blood cultures drawn on the same date. During the same period, there were 59 additional episodes in which a single blood culture had only one of two bottles positive for Bacillus species. Only 2 of 59 such episodes resulted in recurrent bacteremia (3%), as compared with 5 of 17 episodes meeting our case definition (29%) (P = 0.004). In four of five episodes complicated by recurrent bacteremia and in which appropriate antibiotics were used, a Hickman-Broviac catheter was in place and was not removed. We suggest that our case definition permits the differentiation of infection from contamination based on outcome and that patients with Bacillus bacteremia have chronic venous catheters removed as well as receive antibiotic treatment.     

Tentative evidence of AIDS-associated biotype of Mycobacterium kansasii.

Previous studies revealed heterogeneous behavior within the species Mycobacterium kansasii against commercially available DNA probes (Accuprobe M. kansasii culture identification test; Gen-Probe); several isolates, conventionally identified as M. kansasii, failed in fact to hybridize. Looking for a possible association with phenotypic features, we tested a fully characterized panel of 69 clinical isolates of M. kansasii (19 of which were Accuprobe negative) with a semiquantitative micromethod which tests for 19 enzymatic activities (Api Zym; BioMérieux). The strains were from 25 hospitals in 18 Italian towns; 20 isolates came from human immunodeficiency virus type 1-positive patients who fulfilled the Centers for Disease Control criteria for AIDS diagnosis. On the basis of the whole set of phenotypic traits, our strains clustered in two groups, allowing the differentiation of biotypes within the species. There was a perfect association between biotype 2 and hybridization failures with Accuprobe and a very significant association between this novel biotype 2 and AIDS status, which suggests that it differs in virulence.     

Intra-catheter leukocyte culture to monitor hemodialysis catheter colonization. A prospective study to prevent catheter-related bloodstream infections

The most serious problem related to the use of tunneled catheters in hemodialysis is bacteremia. The aim of this study was to detect hemodialysis catheter colonization and, establish a preemptive therapy based on a catheter antibiotic lock in order to prevent development of catheter-related bloodstream infections. During a 24-month period, all patients with tunneled catheters in our hemodialysis unit were evaluated by extracting a through-catheter leukocyte culture every 15 days.There were 28 episodes of catheter colonization occurring in 13 patients (2.2 colonization episodes per 1000 catheter patient-days). At the time of colonization, catheters had been in place for a mean of 562 days (range: 16 to 1475 days). Coagulase negative staphylococci (CNS) were the most common microorganisms to be isolated. A preemptive therapy consisting in teicoplanin locks (10 mg/mL) for 21 days was able to eradicate catheter colonization in 89% of the cases when CNS were isolated. However, relapse of colonization occurred in 61.2% of these cases. The mean duration of catheter use was 239 days (range: 9 to 483 days) after treatment of a colonization episode. The incidence of catheter-related bloodstream infection in our population was 0.78 episodes per 1000 catheter patient-days (IC 95%: 0.374-1.434). This study shows the utility of intra-catheter leukocyte culture for early detection of hemodialysis catheter colonization. Moreover, it establishes that the eradication of biofilm-related CNS is possible without the removal of the catheter, thus enabling a longer catheter lifespan.     

Genotype MRSA, a new genetic test for the rapid identification of staphylococci and detection of mecA gene

Early detection of Staphylococcus methicillin resistance (MR) is essential. However MR determination may be difficult because it is necessary to perform investigation of heterogeneous resistance and low level of resistance and to discriminate between oxacillin resistance and borderline resistance. Several phenotypic methods are recommended but they fail to detect low level of production de PBP2a, the modified Penicillin Binding Protein responsible for MR. Detection of mecA gene, the gene encoding PBP2a, using PCR is considered to be the reference method. We evaluated Genotype MRSA, a new rapid system based on DNA multiplex amplification and further hybridisation, for the identification of staphylococci and detection of the mecA gene. The study was performed on a collection of various Staphylococcus strains (N=30) from clinical human isolates including S. aureus MR and methicillin susceptible (MS), S. epidermidis MR and MS, and other species of coagulase negative Staphylococcus (CNS) MR and MS. For all the strains, the hybridization banding pattern obtained using Genotype MRSA correlated with their expected phenotypic and genotypic characteristics. Genotype MRSA allows the identification of the mecA gene as well as S. aureus and S. epidermidis specific genes. This DNA strip technology based assay can easily be incorporated into routine diagnostics. In addition, the short testing time (less than 2 hours) optimises treatment orientation. Genotype MRSA completely complies with all requirements for a fast, safe, valid and cost-effective MR diagnosis in staphylococci.     

Clinical-epidemiological characteristics and outcome of patients with catheter-related bloodstream infections in Europe (ESGNI-006 Study)

This study analysed 89 episodes of catheter-related bloodstream infection (CR-BSI) occurring during one week in 107 hospitals from 21 European countries (1.02 episodes/1,000 admissions). Patients from European Union (EU) countries had a higher incidence of CR-BSI than patients from non-EU countries (1.55 vs. 0.33/1,000 admissions). Most (67%) catheters were non-tunneled central venous catheters, were in the jugular vein (44%), had been implanted for > 7 days (70%), were made of polyurethane (61%) and were multi-lumen (67%). In 36% of cases, catheters were implanted by physicians other than anaesthetists or surgeons, and 50% were inserted by junior staff.