FRED C. TENOVER,* ROBERT D. ARBEIT, RICHARD V. GOERING, PATRICIA A. MICKELSEN, BARBARA E. MURRAY, DAVID H. PERSING, AND BALA SWAMINATHAN National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333; Veterans Affairs Medical Center, Boston, Massachusetts 02130; Creighton University, Omaha, Nebraska 68178; Stanford University Medical Center, Stanford, California 94305; University of Texas Medical School, Houston, Texas 77030; and Mayo Clinic, Rochester, Minnesota 55905

https://courses.washington.edu/pabio568/files/tenover.pdf

Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing.

Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens.

Diarrheagenic Escherichia coli

Since their initial recognition 20 years ago, Shiga toxin-producing Escherichia coli (STEC) strains have emerged as an important cause of serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic-uremic syndrome. Food-borne outbreaks of STEC disease appear to be increasing and, when mass-produced and mass-distributed foods are concerned, can involve large numbers of people. Development of therapeutic and preventative strategies to combat STEC disease requires a thorough understanding of the mechanisms by which STEC organisms colonize the human intestinal tract and cause local and systemic pathological changes. While our knowledge remains incomplete, recent studies have improved our understanding of these processes, particularly the complex interaction between Shiga toxins and host cells, which is central to the pathogenesis of STEC disease. In addition, several putative accessory virulence factors have been identified and partly characterized. The capacity to limit the scale and severity of STEC disease is also dependent upon rapid and sensitive diagnostic procedures for analysis of human samples and suspect vehicles. The increased application of advanced molecular technologies in clinical laboratories has significantly improved our capacity to diagnose STEC infection early in the course of disease and to detect low levels of environmental contamination. This, in turn, has created a potential window of opportunity for future therapeutic intervention.

Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections

Arias and Murray discuss the factors that may have contributed to the rise of enterococci as nosocomial pathogens, with an emphasis on the epidemiology and pathogenesis of these species and their mechanisms of resistance to the most relevant anti-enterococcal agents used in clinical practice.

The rise of the Enterococcus : beyond vancomycin resistance

Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of stx1, stx2 (including variants of stx2), eaeA, and enterohemorrhagic E. coli hlyA, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the rfb (O-antigen-encoding) regions of E. coli serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic E. coli strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.

Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157

Pulsed-field gel electrophoresis was used to compare 34 isolates of Enterococcus faecium from six different geographic locations. This procedure generated an average of 13 discernible fragment bands per isolate (range, 10 to 19 fragment bands) of 34 to 485 kb. The resulting restriction endonuclease digestion patterns were quite heterogeneous and were able to differentiate 27 of 34 isolates from each other, as defined by one or more mismatched fragment bands. Five patterns were shared by two or more isolates, and each set of isolates with matching patterns (shared pattern) originated in the same medical center, suggesting a common epidemiologic background, including highly penicillin resistant isolates in Richmond and Philadelphia. We conclude that pulsed-field gel electrophoresis of DNA digested with low-frequency-cleavage restriction enzymes offers a relatively simple method of comparing E. faecium for the purpose of epidemiologic study.

DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool.

Two pairs of oligonucleotide primers were designed to amplify fragments of the genes for Shiga-like toxin I (SLT-I) and SLT-II in a single reaction. A 370-bp segment and a 283-bp segment were amplified for SLT-I and SLT-II, respectively. The specificities of the polymerase chain reaction (PCR) amplification products were confirmed by using radioactively labeled oligonucleotide probes. SLT sequences were amplified from DNA isolated from 13 previously characterized enterohemorrhagic Escherichia coli (EHEC) strains. No amplification product was produced by using DNA from 20 non-EHEC strains. As little as one bacterial genome was detectable. PCR was then applied to DNA isolated directly from stool samples. We had to remove inhibitors of PCR that were present in lysates prepared from stool samples before amplification was achieved. First, we evaluated the sensitivity of PCR for the detection of known numbers of EHEC added to normal stools. Second, three children with SLT in their stools were shown to have SLT sequences in their stools by PCR. Two of these children had hemolytic-uremic syndrome, and a third child was asymptomatic. Stool specimens collected from another 26 asymptomatic children were negative by PCR for SLT sequences. PCR can be used to diagnose EHEC infections without prior culture of stool specimens.

Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome.

We examined the in vitro activity of PD 127391, an investigational fluoroquinolone antibacterial agent, against staphylococci (including methicillin-resistant Staphylococcus aureus), enterococci (including beta-lactamase-producing and highly gentamicin-resistant isolates), and streptococci. The compound was active against all organisms tested and compared favorably with antimicrobial agents routinely used to treat infections with these organisms. On the basis of MICs for 90% of the strains tested, PD 127391 was 32-fold more active against all staphylococci, 16-fold more active against methicillin-resistant S. aureus, 8-fold more active against all streptococci, and 4-fold more active against all enterococci than ciprofloxacin. PD 127391 was shown to be more active than sparfloxacin, which in turn was shown to be more active than ciprofloxacin, against these gram-positive cocci. PD 127391 shows promise for the treatment of infections with gram-positive cocci, including organisms which are resistant to other commonly used antimicrobial agents.

/pdf/comparative-in-vitro-activity-of-pd-127391-a-new-2htnkuxsy9.pdf

Comparative in vitro activity of PD 127391, a new fluoroquinolone agent, against susceptible and resistant clinical isolates of gram-positive cocci.

DNA Fingerprinting ofEnterococcus faecium byPulsed-Field Gel Electrophoresis MayBeaUseful Epidemiologic Tool

Pulsed-field gel electrophoresis was used to determine the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium. The size determinations of OG1X, a strain of E. faecalis widely used in many laboratories for genetic studies, using Sma I, Not I, and Sfi I alone or in combination, ranged from 2,750 to 2,761 kb. Using the same enzymes as with OG1X, the size of HH-67, a plasmid-free clinical isolate of E. faecalis, was determined to be 2,170-2,288 kb and the size of JH2-2, an E. faecalis recipient strain, ranged from 2,008 to 2,135 kb. The size range generated for GE-1, a plasmid-free E. faecium strain, with the use of Sma I, Not I, and Apa I was 2,045-2,155 kb. Although OG1X differed in size from the other three enterococci, each individual enterococcal strain generated reproducible results in different experiments. However, for both E. faecalis OG1X and E. faecium GE-1, one of the enzymes used generated a considerably smaller molecular size than that generated by the other two enzymes. The discrepancy was due to visually undiscernible comigrating fragments, and serves to point out a potential source of error if fewer than two enzymes are used to size a genome. The size discrepancies were resolved by digesting individual fragments with a second enzyme. The molecular sizes of these enterococcal strains are larger than that recently reported for Campylobacter, smaller than that of Escherichia coli and Pseudomonas aeruginosa, and similar (OG1X) or smaller (JH2-2, HH67, and GE-1) than the 2,819-kb reported for Streptococcus mutans.

Abraham G. Miranda

Papers

DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool.

Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome.

Comparative in vitro activity of PD 127391, a new fluoroquinolone agent, against susceptible and resistant clinical isolates of gram-positive cocci.

DNA Fingerprinting ofEnterococcus faecium byPulsed-Field Gel Electrophoresis MayBeaUseful Epidemiologic Tool

Determination of the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium.