Serratia marcescens Outbreak in a Neonatal Intensive Care Unit: New Insights from Next-Generation Sequencing Applications.

Serratia marcescens is an environmental bacterium that is commonly associated with outbreaks in neonatal intensive care units (NICUs). Investigations of S. marcescens outbreaks require efficient recovery and typing of clinical and environmental isolates. In this study, we investigated how the use of next-generation sequencing applications, such as bacterial whole-genome sequencing (WGS) and bacterial community profiling, could improve S. marcescens outbreak investigations. Phylogenomic links and potential antibiotic resistance genes and plasmids in S. marcescens isolates were investigated using WGS, while bacterial communities and relative abundances of Serratia in environmental samples were assessed using sequencing of bacterial phylogenetic marker genes (16S rRNA and gyrB genes). Typing results obtained using WGS for the 10 S. marcescens isolates recovered during a NICU outbreak investigation were highly consistent with those obtained using pulsed-field gel electrophoresis (PFGE), the current standard typing method for this bacterium. WGS also allowed the identification of genes associated with antibiotic resistance in all isolates, while no plasmids were detected. Sequencing of the 16S rRNA and gyrB genes both showed greater relative abundances of Serratia at environmental sampling sites that were in close contact with infected babies. Much lower relative abundances of Serratia were observed following disinfection of a room, indicating that the protocol used was efficient. Variations in the bacterial community composition and structure following room disinfection and among sampling sites were also identified through 16S rRNA gene sequencing. Together, results from this study highlight the potential for next-generation sequencing tools to improve and to facilitate outbreak investigations.

Evaluation of molecular typing of foodborne pathogens in European reference laboratories from 2012 to 2013.

In 2012, the European Centre for Disease Prevention and Control (ECDC) initiated external quality assessment (EQA) schemes for molecular typing including the National Public Health Reference Laboratories in Europe. The overall aim for these EQA schemes was to enhance the European surveillance of food-borne pathogens by evaluating and improving the quality and comparability of molecular typing. The EQAs were organised by Statens Serum Institut (SSI) and included Salmonella enterica subsp. enterica, verocytotoxin-producing Escherichia coli (VTEC) and Listeria monocytogenes. Inter-laboratory comparable pulsed-field gel electrophoresis (PFGE) images were obtained from 10 of 17 of the participating laboratories for Listeria, 15 of 25 for Salmonella, but only nine of 20 for VTEC. Most problems were related to PFGE running conditions and/or incorrect use of image acquisition. Analysis of the gels was done in good accordance with the provided guidelines. Furthermore, we assessed the multilocus variable-number tandem repeat analysis (MLVA) scheme for S. Typhimurium. Of 15 laboratories, nine submitted correct results for all analysed strains, and four had difficulties with one strain only. In conclusion, both PFGE and MLVA are prone to variation in quality, and there is therefore a continuous need for standardisation and validation of laboratory performance for molecular typing methods of food-borne pathogens in the human public health sector.

Comparison of pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae.

Biochemical test, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC-PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11­22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216-6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC-PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC-PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods.

PFGE standard operating procedures for Listeria monocytogenes: harmonizing the typing of food and clinical strains in Europe.

Listeria monocytogenes is a foodborne pathogen responsible for a severe disease known as listeriosis. The European Centre for Disease Prevention and Control (ECDC) coordinates a network of national public health laboratories (NPHLs) in charge of typing clinical strains. In food, it is the European Union Reference Laboratory for L. monocytogenes (EURL Lm), which manages a network of National Reference Laboratories (NRLs). A pulsed-field gel electrophoresis (PFGE) standard operating procedure (EURL SOP) has been used routinely at the EURL Lm since 2007. The EURL Lm has recommended that NRLs use the EURL SOP, whereas the Statens Serum Institut (SSI), under contract for ECDC, requested that NPHLs use Halpins' SOP (HSOP) published in 2010 for the PulseNet USA network. An update of Halpins' SOP (uHSOP) was published in 2013. To facilitate the exchange of profiles among human and food European reference laboratories, it is crucial to ensure that the PFGE profiles obtained with these different SOPs are comparable. The aim here was to compare the EURL SOP with HSOP and uHSOP. The panel comprised 114 well-characterized SSI/EURL strains. All were characterized at the EURL using both the EURL SOP and uHSOP. Seventy of the 114 strains were also characterized at the SSI using HSOP. The EURL SOP and uHSOP produced indistinguishable combined (ApaI/AscI) profiles for the 114 strains tested. The EURL SOP and HSOP produced indistinguishable combined profiles for 69 of the 70 strains tested. One strain displayed for the AscI profile an additional low-intensity band at 184 kbp with HSOP. For this strain, SSI and EUR Lm had already observed the same profile from NPHLs and NRLs. However, this deviation is minor as it accounted for about 1% of all the 114 combined profiles. This study should facilitate the exchange of reproducible PFGE profiles among human and food reference laboratories.

High-resolution hydrodynamic chromatographic separation of large DNA using narrow, bare open capillaries: a rapid and economical alternative technology to pulsed-field gel electrophoresis?

A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 µm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10(5) plates for the longest (48.5 kbp) and 3.7 × 10(5) plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed-field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

Pulsed-field gel electrophoresis proficiency testing trials: toward European harmonization of the typing of food and clinical strains of Listeria monocytogenes.

The European Union Reference Laboratory for Listeria monocytogenes (EURL for Lm) coordinates a European network of 35 National Reference Laboratories (NRLs), most of which perform food, environmental, and veterinary Lm strain surveillance in their respective countries. The EURL activities resulted in the recent creation of a database (EURL Lm DB). Typing and related epidemiological data submitted to the EURL Lm DB will be collected and shared by all the NRLs. For a given NRL, the only criterion required in order to submit pulsed-field gel electrophoresis (PFGE) profiles to the database was the successful participation with at least one EURL PFGE and PFGE profile interpretation Proficiency Testing (PT) trial. In this context, the EURL organized a PT trial in 2012 to evaluate the NRL's ability to perform PFGE and profile interpretation. A total of 18 NRLs took part in this study. Upon request from the Food- and Waterborne Diseases and Zoonoses Programme of the European Centre for Disease Prevention and Control, 10 National Public Health Reference Laboratories (NPHLs) also took part in this PT trial. Of the 28 participating laboratories, 16 obtained results classified as "good" or "satisfactory." These 16 laboratories included 10 NRLs (56%) and 6 NPHLs (60%). Of the 22 NRLs and NHPLs that participated in the part of the PT trial related to PFGE profile interpretation, 11 laboratories obtained good results. These 11 laboratories included eight NRLs, which therefore can now submit profiles to the EURL Lm DB. This PT trial provided a valuable opportunity to facilitate and to stimulate the sharing of reproducible PFGE profiles between human and food reference laboratories.

Pulsed-field gel electrophoresis, conventional, and molecular serotyping of Listeria monocytogenes from food proficiency testing trials toward an harmonization of subtyping at European level.

The European Union Reference Laboratory for Listeria monocytogenes (EURL for L. monocytogenes) coordinates a European network of 29 National Reference Laboratories (NRLs). Depending on a national decision, NRLs undertake food, environmental, and veterinary L. monocytogenes strain surveillance in their respective countries. In the framework of the PulseNet Europe network, two pulsed-field gel electrophoresis (PFGE) subtyping proficiency testing (PT) trials were carried out in 2003 and 2006. The obtained data showed that PFGE profiles can be compared and exchanged between laboratories. However, no further PT trial had been performed since 2006. In this context, two PT trials were organized by the EURL to evaluate the ability of NRLs to perform conventional serotyping, molecular serotyping and PFGE subtyping. Eleven well-characterized isolates of L. monocytogenes were used: six and nine isolates were tested in 2009 and 2010, respectively. Three isolates were repeated between the two studies. In the 2010 panel, a strain was tested in duplicate, and two strains were related to the same epidemiological group. The strains were analyzed blind in different laboratories (17 in 2009 and 25 in 2010) using (1) their own in-house method for serotyping methods and (2) standardized protocols based on the PulseNet protocol for PFGE. For conventional serotyping, 86.0% in 2009 and 91.0% in 2010 of the serotypes obtained were in agreement with the EURL data. For molecular serotyping, 93.5% of the results in 2009 and 95.2% in 2010 matched the EURL data. For PFGE, 68.9% in 2009 and 81.7% of the combined AscI/ApaI profiles were indistinguishable from the EURL reference profiles. The variations observed could be attributed to slight standardization defaults or, in a few cases, to a failure in DNA extraction. These PT trials provided a valuable opportunity to improve the subtyping ability of NRLs and facilitate exchanges of subtyping data in the future.

Optimization of pulsed-field gel electrophoresis protocols for Salmonella Paratyphi A subtyping.

Salmonella enterica serovar Paratyphi A infection has caused public health problems in some countries in recent years. Pulsed-field gel electrophoresis (PFGE) has been used for the subtyping and epidemiological investigations of some serotypes of Salmonella, mainly in outbreaks caused by non-typhoidal Salmonella. In this study, different restriction endonucleases and electrophoresis parameters were compared for the PFGE subtyping by using Salmonella Paratyphi A strain panels. Two protocols for the enzymes SpeI and XbaI showed higher discriminatory power, which may facilitate epidemiological analysis for more accurate case definition, and clonality study of Salmonella Paratyphi A.

Re-evaluation, optimization, and multilaboratory validation of the PulseNet-standardized pulsed-field gel electrophoresis protocol for Listeria monocytogenes.

The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms.

Comparison of pulsed-field gel electrophoresis and whole-genome-sequencing-based typing confirms the accuracy of pulsed-field gel electrophoresis for the investigation of local Pseudomonas aeruginosa outbreaks.

AIM: To determine whether pulsed-field gel electrophoresis (PFGE) accurately recognizes isolates belonging to clusters defined by techniques based on whole-genome sequencing (WGS) using Pseudomonas aeruginosa as a model. METHODS: We selected 65 isolates of ST395 P. aeruginosa isolated in seven European hospitals between 1998 and 2012. Isolates were typed by PFGE and sequenced by WGS. A core genome multi-locus sequence typing (cgMLST) analysis based on 3831 genes was performed with a homemade pipeline. FINDINGS: PFGE identified eight pulsotypes and cgMLST differentiated nine clusters and nine singletons. Five cgMLST clusters and pulsotypes (31/65 isolates) coincided perfectly. Isolates without evident epidemiological links grouped by PFGE were separated by cgMLST (16/65 isolates) differentiating cities, suggesting that PFGE should be kept for the investigation of local outbreaks. Importantly, hypermutator isolates still shared the pulsotype with their parents (16/65 isolates), whereas they were not recognized by cgMLST. This shows that PFGE was less affected than WGS-based typing by the accelerated genetic drift that occurs in epidemic P. aeruginosa. CONCLUSIONS: although WGS-based typing has logically become the new reference standard, we show here that the PFGE can be used with confidence for the investigation of local outbreaks caused by P. aeruginosa.

The optimization of a rapid pulsed-field gel electrophoresis protocol for the typing of Acinetobacter baumannii, Escherichia coli and Klebsiella spp.

Pulsed-field gel electrophoresis (PFGE) is the most common genotyping method used for the typing of a number of bacterial species. Generally, investigators use their own custom-developed protocol, but a standardized PFGE protocol would allow the comparison of typing results between laboratories and the tracing of strains around the country. In the present study, we optimized a PFGE protocol for subtyping of Acinetobacter baumannii, Escherichia coli and Klebsiella spp., which are commonly isolated from nosocomial infections in many hospitals. Reproducibility of our PFGE procedure was studied three times at 2- to 3-week intervals. Epidemiological concordance of the optimized PFGE procedure was tested on seven isolates of A. baumannii from a previous outbreak and seven A. baumannii isolates randomly selected among the clinical isolates. The optimized PFGE procedure was evaluated on a total of 174 clinical isolates including 62 A. baumannii, 50 E. coli, and 62 Klebsiella spp. The inter-laboratory reproducibility of the optimized protocol was tested at four laboratories. The optimized procedure is completed in 28 h after culturing. It is likely to be cost-effective, due to the reduction in the time, reagent volume and enzyme concentration needed. The procedure showed high concordance with epidemiological data. There were no non-typeable isolates among the tested bacteria. It is reproducible and versatile. This protocol can be used to identify outbreaks and monitor the spreading rate of nosocomial infections caused by the tested bacterial isolates. Furthermore, due to its high intra- and inter-laboratory reproducibility, the protocol has the potential to be useful for comparing PFGE fingerprinting profiles of the isolates from different settings.

Evaluation and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping Vibrio parahaemolyticus: an international multicenter collaborative study.

The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.

Evaluation of restriction enzymes for standardizing pulsed-field gel electrophoresis protocol for rapid subtyping of Vibrio parahaemolyticus.

We evaluated the cost-effectiveness of the restriction enzymes with rare-cutting sites in the genome of Vibrio parahaemolyticus RIMD 2210633 for pulsed-field gel electrophoresis (PFGE) analysis. The evaluation indicated that PFGE with both NotI and SfiI was discriminatory, but NotI was more cost-effective. Based on the results of this study, we suggest using NotI and SfiI as the 1st and the 2nd restriction enzyme for standardizing the PulseNet PFGE protocol for molecular subtyping and global surveillance of V. parahaemolyticus.

Improvement and validation of RAPD in combination with PFGE analysis of Salmonella enterica ssp. enterica serovar Senftenberg strains isolated from feed mills.

In 1995 and 1996 a Swedish feed mill had problems due to a persistent contamination of Salmonella enterica spp. enterica serovar Senftenberg that was difficult to eliminate. Forty-eight strains isolated from the feed mill, together with unrelated strains included to evaluate the discriminatory power and reproducibility, were analysed by pulsed-field gel electrophoresis (PFGE). The source of contamination in the feed mill was identified and preventative measures were taken, that led to a resolution of the problem. A previously developed randomly amplified polymorphic DNA (RAPD) protocol was used, to evaluate a rapid and low-cost alternative to PFGE typing. The use of the alternative thermostable DNA polymerase Tth was shown to increase the reproducibility of the RAPD analysis. The reproducibility, in terms of Pearson's and Dice's similarity coefficients for duplicate runs, increased from 72.0 +/- 16.9% and 72.3 +/- 12.9% for Taq to 91.6 +/- 7.5% and 90.9 +/- 5.3% for the fingerprints obtained for the RAPD method employing Tth DNA polymerase. Simpson's index of diversity was calculated and found to be 0.580 for RAPD and 0.896 for PFGE. All of the seven RAPD types could be subdivided into one or more PFGE types, whereas none of the 22 PFGE types was divided into more than one RAPD type. RAPD provides a simple, rapid and powerful screening method that can be used to initially select isolates for further analysis by PFGE.

Genomic characterization of oxacillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from Brazilian medical centres.

Studies on the genetic diversity of oxacillin-resistant coagulase-negative staphylococcal (CNS) isolates are important for the control and prevention of infections. The present study evaluated the clonal diversity of oxacillin-resistant Staphylococcus epidermidis (ORSE) and Staphylococcus haemolyticus (ORSH) strains, isolated from patients in nine Brazilian medical centres by using pulsed-field gel electrophoresis (PFGE) after digestion of bacterial DNA using SmaI. PFGE analysis of ORSE (N=44) and ORSH (N=25) strains showed the presence of 29 restriction profiles clustered in 16 PFGE types, and 21 distinct profiles in 15 PFGE types, respectively, indicating a large genetic diversity among isolates of both of these species. Among the ORSE isolates, 23 (52%) strains belonged to two predominant PFGE types (named A and B), which were observed in most of the hospitals assessed, indicating the spread of these PFGE types in hospitals located in Rio de Janeiro. The spread of PFGE types of ORSH was also detected in some of the hospitals investigated. The results show that PFGE is a suitable tool for epidemiological studies of oxacillin-resistant CNS, and can be used as a basis for infection control procedures for these multiresistant organisms.

Comparative genomic hybridisation using a proximal 17p BAC/PAC array detects rearrangements responsible for four genomic disorders.

BACKGROUND: Proximal chromosome 17p is a region rich in low copy repeats (LCRs) and prone to chromosomal rearrangements. Four genomic disorders map within the interval 17p11-p12: Charcot-Marie-Tooth disease type 1A, hereditary neuropathy with liability to pressure palsies, Smith-Magenis syndrome, and dup(17)(p11.2p11.2) syndrome. While 80-90% or more of the rearrangements resulting in each disorder are recurrent, several non-recurrent deletions or duplications of varying sizes within proximal 17p also have been characterised using fluorescence in situ hybridisation (FISH). METHODS: A BAC/PAC array based comparative genomic hybridisation (array-CGH) method was tested for its ability to detect these genomic dosage differences and map breakpoints in 25 patients with recurrent and non-recurrent rearrangements. RESULTS: Array-CGH detected the dosage imbalances resulting from either deletion or duplication in all the samples examined. The array-CGH approach, in combination with a dependent statistical inference method, mapped 45/46 (97.8%) of the analysed breakpoints to within one overlapping BAC/PAC clone, compared with determinations done independently by FISH. Several clones within the array that contained large LCRs did not have an adverse effect on the interpretation of the array-CGH data. CONCLUSIONS: Array-CGH is an accurate and sensitive method for detecting genomic dosage differences and identifying rearrangement breakpoints, even in LCR-rich regions of the genome.

Evaluation of pulsed-field gel electrophoresis analysis performed at selected prefectural institutes of public health for use in PulseNet Japan.

In order to evaluate reliability of pulsed-field gel electrophoresis (PFGE) analysis performed at different prefectural public health institutes (PHIs) for use in the PulseNet Japan surveillance system to detect enterohemorrhagic Escherichia coli O157, we compared the results of PFGE-typing of 14 selected strains of O157 performed at 8 selected PHIs to evaluate the reliability of different experimental protocols used in these PHIs. PFGE was performed for 14 strains for which there were 14 PFGE types in 3 PHIs, and 13 PFGE types in 5 PHIs by using their own protocols and/or those of the National Institute of Infectious Diseases (NIID). PFGE fingerprints from 5 out of the 8 PHIs were successfully genotyped for all of the 14 strains. A PFGE fingerprint from one PHI was successfully genotyped when the NIID pulsing protocol was used, but was not genotyped when the PHI's own protocols were used. PFGE fingerprints from 2 PHIs failed to be genotyped for one each of the strains. The cause of this genotyping failure was considered to be inappropriate PFGE pulsing protocols or inadequate digestion of chromosomal DNA. These results suggest that PFGE protocols should be standardized for the establishment of PulseNet Japan.

Method for optimizing pulsed-field gel electrophoresis banding pattern data.

The genomic DNA of 47 strains of TSST-1 toxin-producing Staphylococcus aureus were cleaved with SmaI restriction endonuclease and resolved in an agarose gel by pulsed-field gel electrophoresis (PFGE). An algorithm was designed to standardize the band weights or brightness (trace quantity) produced to a bounded region between 0 and 1 regardless of DNA fragment size while simultaneously reducing gel-to-gel variability. The algorithm allows for classification of isolates by band intensity as well as DNA mobility without a numerical hierarchy of band intensity that is caused by ranging DNA fragment lengths. On analysis many isolates were classified as separate entities on the basis of DNA co-migration only. Isolates differing by only DNA co-migration were subjected to a second digestion with restriction enzyme SacII. These isolates were characterized similarly to the standardized trace quantity analysis of SmaI PFGE patterns. The standardization method proposed in this article permits characterization of isolates on the basis of band differences, regardless of DNA co-migration, thus increasing the discriminatory power (0.79 to 0.89) of PFGE by increasing band-associated information. An established unbiased approach to the partitioning of data were also explored.

Monitoring persistence of coagulase-negative staphylococci in a hematology department using phenotypic and genotypic strategies.

OBJECTIVE: To determine persistence of coagulase-negative staphylococci (CNS) on a hematology-oncology ward and to determine the value of phenotypic and genotypic procedures for establishing clonality among CNS isolates. DESIGN: Strains of CNS isolated from bacteremic patients (n = 139) were typed by biochemical reactivity, antibiotic susceptibility, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), and arbitrary primed polymerase chain reaction (AP PCR). Coagulase-negative staphylococci were subgrouped in a random collection (n = 20) used for the evaluation of the typing procedures and a collection of 119 CNS isolates from hematologic patients displaying multiple bacteremic episodes. RESULTS: Analysis of the reference collection demonstrated the usefulness of the DNA typing procedures, indicating that AP PCR and PFGE can be used for epidemiologic typing of CNS in a concordant fashion. Certain strains appeared to be permanent colonizers of the hematology ward or ward-related personnel. In individual patients, persistent colonization by a single type was demonstrated. However, a number of patients also experienced bacteremic episodes caused by CNS belonging to different types. CONCLUSION: We conclude that monitoring of CNS infections on a hematology ward by various genotypic techniques provides insight into nosocomial epidemiology and elucidates the complexity of the infections taking place. DNA typing is preferred over phenotypic procedures and can identify persistent CNS strains in a given location.