Development of a universal double-digest RAD sequencing approach for a group of nonmodel, ecologically and economically important insect and fish taxa.

The generation of genome-scale data is critical for a wide range of questions in basic biology using model organisms, but also in questions of applied biology in nonmodel organisms (agriculture, natural resources, conservation and public health biology). Using a genome-scale approach on a diverse group of nonmodel organisms and with the goal of lowering costs of the method, we modified a multiplexed, high-throughput genomic scan technique utilizing two restriction enzymes. We analysed several pairs of restriction enzymes and completed double-digestion RAD sequencing libraries for nine different species and five genera of insects and fish. We found one particular enzyme pair produced consistently higher number of sequence-able fragments across all nine species. Building libraries off this enzyme pair, we found a range of usable SNPs between 4000 and 37 000 SNPS per species and we found a greater number of usable SNPs using reference genomes than de novo pipelines in STACKS. We also found fewer reads in the Read 2 fragments from the paired-end Illumina Hiseq run. Overall, the results of this study provide empirical evidence of the utility of this method for producing consistent data for diverse nonmodel species and suggest specific considerations for sequencing analysis strategies.

Masking in waved-2 mice: EGF receptor control of locomotion questioned.

It has been suggested that epidermal growth factors (EGF) are responsible for the inhibition of locomotion by light (i.e., masking) in nocturnal rodents (Kramer et al., 2001). The poor masking response of waved-2 (Egfr(wa2)) mutant mice, with reduced EGF receptor activity, was adduced in support of this idea. In the present work, we studied the responses to light over a large range in illumination levels, in a variety of tests, with pulses of light and with ultradian light-dark cycles in Egfr(wa2) mutant mice. No evidence suggested that normal functioning of epidermal growth factor receptors was required, or even involved, in masking.

Penicillin-resistant Streptococcus pneumoniae in metropolitan New York hospitals: case control study and molecular typing of resistant isolates.

During the 4-month period from January to April, 1998, 476 patients with Streptococcus pneumoniae infections were detected in 12 metropolitan New York hospitals and 112 penicillin-resistant (PRP) isolates (24%) were identified in 11 institutions. A case control study of 100 patients with penicillin-resistant and susceptible pneumococci from four of the widely dispersed hospitals revealed a high incidence of underlying medical illnesses in adult patients (74%), a preponderance of patients with pneumonia (63%), and a majority of patients who had underlying risk factors for pneumonia or invasive disease (51%). In this limited case control study, no difference was noted between cases and controls regarding known risk factors for penicillin-resistant pneumococcal infections. The percentage of single-patient PRP isolates varied among individual hospitals but the mean percentages of PRP from the four participating University Medical Centers and seven community hospitals were similar: 26% and 22% respectively. By E-test, 60% and 26% were high-level penicillin and ceftriaxone resistant, respectively. Pulsed-field gel electrophoresis identified 26 chromosomal macrorestriction patterns among the 103 PRP isolates available for analysis, but almost half (50 isolates or 48%) of these belong to two drug-resistant internationally spread clones, SP(23)-1 and SP(9/14)-3, that were detected in all hospitals and were recovered from invasive and noninvasive sites in both children and adults.

Cost of hospitalization for and risk factors associated with vancomycin-resistant Enterococcus faecium infection and colonization.

The increase in costs of hospitalization for patients with drug-resistant infection may be associated with drug resistance itself or with the severity of the underlying illness that predisposes patients to acquire the drug-resistant infection. To address this issue, risk factors and cost of hospitalization were compared for patients infected or colonized with vancomycin-susceptible Enterococcus faecium (VSEF) or vancomycin-resistant E. faecium (VREF) in a large tertiary-care hospital in New York City. From January 1995 through December 1996, 157 patients with VSEF and 262 patients with VREF were identified. CMI (case-mix index) was assigned to each patient as a measure of severity of illness, with a CMI of 1 considered to represent illness of average severity. For all patients who were assigned a CMI of <3, the cost per day of hospitalization for patients with VREF was significantly greater than that for patients with VSEF. However, for patients with a CMI of >3, there was no difference between cost of hospitalization for patients with VREF and that for patients with VSEF. These observations indicate that, although vancomycin resistance is associated with an increased cost of hospitalization for less severely ill patients with VREF, patients with severe underlying illness, regardless of vancomycin resistance, incur similar hospitalization costs.

Distribution of methicillin-resistant Staphylococcus aureus clones among health care facilities in Connecticut, New Jersey, and Pennsylvania. .

A previous surveillance study conducted in 12 hospitals in New York City in 1996 identified a unique multidrug-resistant genetic lineage of methicillin-resistant Staphylococcus aureus (MRSA) that was widespread and accounted for as much as 42% of all the MRSA isolates. The purpose of the study described here was to determine possible geographic spread of this New York clone of MRSA to neighboring states. Single-patient MRSA isolates (258) from 29 health care facilities in Connecticut (CT), New Jersey (NJ), and Pennsylvania (PA) were collected during the calendar year 1998. DNA typing, consisting of fingerprinting of chromosomal macrorestriction patterns generated by SmaI digestion followed by pulsed-field gel electrophoresis (PFGE), identified 22 patterns. PFGE type A, closely related to the PFGE type of the previously identified New York clone, accounted for 154 (60%) of 258 isolates. The clone was detected in all facilities, was predominant in 19 of the 29 health care centers, and accounted for 92% of the MRSA isolates collected in PA. The overwhelming majority of MRSA with PFGE type A was also resistant to erythromycin, ciprofloxacin, and clindamycin. One of the two most common PFGE subtypes detected in the three states sampled (PFGE subtype A1) had an identical PFGE pattern to that of the previously described vancomycin-resistant strain of S. aureus (VISA) recently detected in a hospital in Westchester, NY. The second most frequent MRSA clone with PFGE type E and accounting for 26% (68/258 isolates), also described earlier in the 12 New York City hospitals, was resistant not only to erythromycin, ciprofloxacin, and clindamycin, but also to gentamicin and sulfamethoxazole-trimethoprim as well. The unique multidrug resistance pattern of this second clone and its geographic distribution accounted for the differences observed in the frequency of multidrug resistance among MRSA isolates recovered in the three states. The pandemic Iberian clone recently detected in New York City was not detected among the 258 MRSA isolates recovered in CT, NJ, and PA.

Practical approach to the identification of clinically relevant Enterococcus species.

Enterococci have become important nosocomial pathogens, with Enterococcus faecalis and then Enterococcus faecium predominating. Because of the emergence of glycopeptide (vancomycin and teicoplanin) resistance in enterococci, laboratories have been required to screen for resistant strains and to identify them to the species level. This has resulted in the need for accurate identification of species less commonly associated with clinical infections, such as Enterococcus casseliflavus and Enterococcus gallinarum, which are inherently resistant to the glycopeptides. Studies evaluating commonly used commercial identification systems, have found error rates for enterococcal species identification of 2-21% for E. faecalis, 5-9% for E. faecium, and 14-79% for other species. Reporting errors may have adverse effects on the management of clinical infections, as well as in the control of multidrug-resistant strain outbreaks. The purpose of this document is to present a simplified approach to the identification of Enterococcus species that uses a combination of rapid, readily available, and inexpensive tests.

Heterogeneously vancomycin-resistant Staphylococcus epidermidis strain causing recurrent peritonitis in a dialysis patient during vancomycin therapy.

Methicillin-resistant Staphylococcus epidermidis (MRSE) was recovered over a 2-month period from the dialysis fluid of a peritoneal dialysis (PD) patient who experienced recurrent episodes of peritonitis during therapeutic and prophylactic use of vancomycin. Characterization of five consecutive MRSE isolates by molecular and microbiological methods showed that they were representatives of a single strain, had reduced susceptibility to vancomycin, did not react with DNA probes specific for the enterococcal vanA or vanB gene, and showed characteristics reminiscent of the properties of a recently described vancomycin-resistant laboratory mutant of Staphylococcus aureus. Cultures of these MRSE isolates were heterogeneous: they contained-with a frequency of 10(-4) to 10(-5)-bacteria for which vancomycin MICs were high (25 to 50 microg/ml) which could easily be selected to "take over" the cultures by using vancomycin selection in the laboratory. In contrast, the five consecutive MRSE isolates recovered from the PD patient during virtually continuous vancomycin therapy showed no indication for a similar enrichment of more resistant subpopulations, suggesting the existence of an "occult" infection site in the patient (presumably at the catheter exit site) which was not accessible to the antibiotic.

Outbreak in a New York City teaching hospital burn center caused by the Iberian epidemic clone of MRSA.

During an 18-month period in a burn center (January 1995 through June 1996), 109 single-patient MRSA isolates were identified and 102 isolates (94%) were available for DNA fingerprinting. Ninety-nine isolates (97%) carried the mecA polymorph I and Tn554 type E. Pulsed-field electrophoresis (PFGE) identified 8 patterns, of which 60 isolates were of pattern F2. The I:E:F clonal type and a stable drug multidrug resistant phenotype (sensitivity only to trimethoprim/sulfamethoxazole and vancomycin) indicated that these isolates were closely related to the Iberian clone of MRSA, which is widely spread in Europe. The initial source of I:E:F isolates was sputum 49%, blood 23%, wound 16%, urine 7%, and intravascular catheter tip 5%. Fifty-four percent of patients had smoke inhalation injury, and 51/53 required intubation or tracheostomy. Forty-three isolates were considered invasive (positive blood culture). The overall mortality was 30%. Despite infection control measures, the I:E:F clone continued to be recovered from patients during the 18 months of study. This outbreak is the first known report of the Iberian MRSA clone in the United States.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in 12 New York hospitals. MRSA Collaborative Study Group.

Consecutive single-patient methicillin-resistant Staphylococcus aureus (MRSA) isolates (270) from 12 hospitals (8217 beds) in metropolitan New York City were collected during May 1996. In 11 of 12 hospitals, MRSA was most frequent in the general medical services. DNA typing ("fingerprinting") revealed that mecA:Tn554:PFGE (pulsed-field gel electrophoresis) type I:A:A accounted for 113 (42%) of 270 isolates, was detected in all hospitals, and was the predominant clone in 9. Thirteen of 15 I:E:F isolates were from 1 hospital, and the remaining 2 were from another hospital of the same health system. Type V:NH:E was isolated from 22 (79%) of the 28 patients with AIDS, including 8 of 9 patients from an additional hospital. Subtype V:NH:E2 was recovered from 11 patients, 9 of whom had AIDS, including all 5 AIDS patients from one floor of a nursing home affiliated with a third hospital. By using both mecA:Tn554 probes and PFGE, MRSA clusters and outbreaks may be detected and provide a rationale for appropriate infection control intervention.

Studies of the continuing susceptibility of group A streptococcal strains to penicillin during eight decades.

BACKGROUND: In view of the widespread use of penicillin for >50 years for the treatment of group A streptococcal infections, we examined the question of whether there has been a change in susceptibility to penicillin in group A streptococcal strains collected during a span of 80 years (1917 to 1997). METHODS: One hundred thirty-three group A streptococcal strains collected during 80 years were tested for changes in penicillin susceptibility. Three tests were used: (1) the microtiter broth minimal inhibitory concentration (MIC); (2) the minimal bactericidal concentration (MBC); and (3) the penicillin E strip MIC. RESULTS: The results indicate there has been no change in the susceptibility to penicillin in these group A streptococci during the past 80 years. The microtiter broth MIC90 for the oldest strains (0.032 microg/ml) was not significantly different from those collected most recently (0.032 microg/ml); there is no statistical difference between the raw MIC data for the four collection periods (P=0.468, analysis of variance on ranks). CONCLUSIONS: There has been no change in the susceptibility of group A streptococci during this time in spite of well-documented cases of penicillin resistance in other Gram-positive organisms and despite recognized resistance of group A streptococci to other antibiotics.

Why have group A streptococci remained susceptible to penicillin? Report on a symposium.

In spite of 50 years of extensive use of penicillin, group A streptococci remain exquisitely susceptible to this antibiotic. This observation that continuing susceptibility has occurred despite the development of resistance to other antimicrobial agents prompted a day-long meeting at Rockefeller University (New York) in October 1996. Among the most likely explanations for this remarkable state of continued susceptibility to penicillin are that beta-lactamase may not be expressed or may be toxic to the organism and/or that low-affinity penicillin-binding proteins either are not expressed or render organisms nonviable. Other potential explanations are that circumstances favorable for the development of resistance have not yet occurred and/or that there are inefficient mechanisms for or barriers to genetic transfer. Recommended future actions include (1) additional laboratory investigations of gene transfer, penicillin-binding proteins, virulence factors, and homeologous recombination and mismatch repair; (2) increased surveillance for the development of penicillin resistance; (3) application of bioinformatics to analyze streptococcal genome sequences; and (4) development of vaccines and novel antimicrobial agents. Thus far the susceptibility of group A streptococci to penicillin has not been a major clinical or epidemiological problem. A similar observation, however, could have been made decades ago about Streptococcus pneumoniae. It is therefore vital for the scientific community to closely examine why penicillin has remained uniformly highly active against group A streptococci in order to maintain this desirable state.

Chlorhexidine gluconate (CHG) activity against clinical isolates of vancomycin-resistant Enterococcus faecium (VREF) and the effects of moisturizing agents on CHG residue accumulation on the skin.

The effectiveness of skin decontamination by chlorhexidine gluconate (CHG) in the presence of commonly-used skin moisturizing lotions was evaluated using vancomycin-resistant Enterococcus faecium (VREF) as a representative nosocomial pathogen. Anti-bacterial efficacy was determined in vitro using pigskin preparations inoculated with five VREF clinical isolates to evaluate Calgon Vestal 2 and 4% (by weight) CHG solutions in comparison with Hibiclens Antiseptic Antimicrobial Cleaner (4% CHG solution). Control inocula were determined for each experiment from recovery of VREF harvested directly from the surface of each control piece of skin. These CHG formulations were evaluated in the presence and absence of Calgon Vestal 'Lotion Soft Skin Conditioner' (LSSC) to determine potential interactions of CHG with LSSC, and also with ¿Vaseline Intensive Care' lotion as a CHG-deactivating agent. The 2% Calgon Vestal CHG alone reduced VREF 10(2)-10(3)-fold, as well as 10(3)-10(4)-fold when LSSC was present, and was as efficacious as either 4% CHG solution when these were tested in the presence of LSSC. Four percent Calgon Vestal CHG produced reductions of 10(3)-10(5)-fold with or without LSSC present. Conversely, ¿Hibiclens' showed similar reductions in the presence of LSSC to that for the Calgon Vestal 4% CHG, but only a 10(1)-10(3)-fold reduction without LSSC. ¿Vaseline Intensive Care' lotion completely inactivated the VREF-killing effects for all of the CHG formulations tested, while LSSC and ¿Vaseline Intensive Care' lotion both showed minimal activity alone against these VREF isolates. These results indicate that the Calgon Vestal 2% CHG solution is as effective against VREF, even in the presence of LSSC, as either the 4% Calgon Vestal or Hibiclens 4% CHG formulations; the use of this lower concentration of CHG may be associated with less irritation, particularly with concomitant use of LSSC.

Risk factors associated with vancomycin-resistant Enterococcus faecium infection or colonization in 145 matched case patients and control patients.

Risk factors and mortality associated with vancomycin-resistant Enterococcus faecium (VREF) infection or colonization were examined at a tertiary care hospital by comparing 145 patients who had VREF isolates (cases) to 145 patients with vancomycin-susceptible Enterococcus faecium (VSEF) isolates (controls). The number of deaths per 100 person-days of hospitalization after diagnosis did not differ significantly between VREF patients (1.2) and VSEF patients (0.8). Multivariate analyses found that the duration of hospitalization ( > or = 7 days), intrahospital transfer between floors, use of antimicrobials (i.e., vancomycin and third-generation cephalosporins), and duration of vancomycin use ( > or = 7 days) was independently associated with VREF infection or colonization. This study, which has a large sample size, confirms some earlier observations regarding risks for VREF infection or colonization and identifies factors that may be potentially exploited to develop interventional strategies for the control of this emerging nosocomial problem.

Multiplicity of genetic backgrounds among vancomycin-resistant Enterococcus faecium isolates recovered from an outbreak in a New York City hospital.

A total of 182 vancomycin-resistant Enterococcus faecium and 6 Enterococcus faecalis inpatient isolates recovered during a 2-year period (1990-1992) in a New York City hospital were analyzed by molecular fingerprinting techniques, pulsed-field gel electrophoresis (PFGE), of chromosomal SmaI digests combined with Southern hybridization using vanA and vanB2-specific DNA probes. Of the 180 isolates hybridizing with these probes, 153 carried the vanA and 27 the vanB gene. As many as 21 different PFGE types and a total of 54 subtypes were identified among the isolates, and the size of vanA and vanB-hybridizing DNA fragments also showed a wide range of sizes, from about 37 to over 280 kb (in vanA) or 140 kb (in vanB), suggesting extensive recombination, including chromosomal integration, of the resistance genes in the isolates. Close to one-third, 46, of the 148 isolates from 1992 belonged to two closely related PFGE subtype variants, each of which carried a 48 kb vanA hybridizing DNA fragment. Spread of this clone appears to be mainly responsible for the substantial increase in the prevalence of vancomycin-resistant E. faecium in early 1992.

Testing the efficacy of a molecular surveillance network: methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) genotypes in six hospitals in the metropolitan New York City area. The BARG Initiative Pilot Study Group. Bacterial Antibiotic Resistance Group.

Molecular fingerprinting techniques are rapidly becoming indispensable tools for hospital epidemiology. On the other hand, the relative complexity and unfamiliarity of these techniques to most hospital diagnostic laboratories limit their usefulness. In an attempt to provide a solution for this dilemma, we tested the feasibility and efficacy of a cooperative venture in which molecular typing of isolates recovered from patients in six hospitals was performed at two microbiology research laboratories with expertise in these techniques. In a small preliminary study, 30 methicillin-resistant Staphylococcus aureus (MRSA) and 30 vancomycin-resistant Enterococcus faecium (VREF) isolates were collected over a 3-week period from six hospitals in the metropolitan New York area and transported to the Laboratory of Microbiology at The Rockefeller University during the summer months of 1994. Nineteen of the 27 confirmed MRSA isolates were closely related strains carrying the same mecA and the same Tn554 polymorphs in a pulsed-field gel electrophoresis (PFGE) background represented by closely related subtypes of a single pattern, indicating the wide distribution of this MRSA clone among the participating hospitals. Typing of the same 27 MRSA isolates was also performed at the Tuberculosis Center of the Public Health Research Institute and identical results were obtained. The 29 confirmed VREF isolates were highly heterogeneous and belonged to as many as 23 distinct clonal types as defined by PFGE patterns and probing with vanA. Characterization of the 60 isolates by these methods was completed in one month of full-time effort by a single experienced laboratory assistant guided by a doctoral-level expert in molecular fingerprinting techniques. The collection of samples for both MRSA and VREF was not intended to address epidemiological questions but to determine the feasibility of a multicenter study. On the basis of our preliminary findings we are encouraged that a larger cooperative effort is possible and with the correct sampling method we believe that epidemiological and surveillance studies could be accomplished that would provide a tracking system to assist hospitals, clinics, and chronic care facilities in controlling the spread of multidrug-resistant pathogens.

Association of penicillin-resistant pneumococci with residence in a pediatric chronic care facility.

Risk factors for the acquisition of penicillin-resistant pneumococci (PRP) were analyzed at a university hospital in New York City. Patients with PRP and control patients with penicillin-sensitive pneumococcal infections were compared. In 1994, 24 (21%) of 113 patients with Streptococcus pneumoniae infections had PRP; 13 PRP isolates were from children and 11 from adults. Only white race (P < .05) and residence in a pediatric chronic care facility (P < .05) were significantly associated with penicillin resistance. An investigation at one chronic care facility revealed that 33% of children (17/52) had PRP colonization. Fourteen of the 17 PRP isolates were also resistant to ceftriaxone. Prior antibiotic use and specifically beta-lactam use were associated with penicillin resistance. All typeable PRP isolates were multidrug-resistant serotype 23. Pediatric residents in chronic care facilities may be an important reservoir of PRP and may serve as a source of PRP transmission when they are transferred to acute care hospitals.

Clinical and microbiological characteristics of severe group A streptococcus infections and streptococcal toxic shock syndrome.

We have monitored all cases of invasive group A streptococcus (GAS) infection that have occurred at the New York Hospital (New York) since 1989. Five cases of GAS infection and shock were identified between 1990 and 1991, and an additional case was recently identified at an affiliated hospital. Five of the six patients met the case definition for streptococcal toxic shock syndrome (strep TSS). Three were bacteremic, and four had aggressive soft-tissue infections. Patients with shock, for whom the mortality was higher, had fewer underlying illnesses than did patients who had GAS bacteremia without shock. Although the M1 serotype and production of streptococcal pyrogenic exotoxin A were more common in patients with GAS infection and shock, several patients with strep TSS were infected with a nontypable strain of GAS that produced only streptococcal pyrogenic exotoxin B. In addition, we observed a distinctive early hemodynamic profile for patients with strep TSS that was unlike that for patients who had typical gram-negative septic shock; this profile was consistent with toxic cardiomyopathy (i.e., relatively low cardiac output, low-to-normal systemic vascular resistance, and striking reduction in ventricular performance.