Efrain M. Ribot

Ceftriaxone-Resistant Salmonella Infection Acquired by a Child from Cattle

BACKGROUND The emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. Resistance to ceftriaxone and the fluoroquinolones, which are used to treat invasive salmonella infections, is rare in the United States. We analyzed the molecular characteristics of a ceftriaxone-resistant strain of Salmonella enterica serotype typhimurium isolated from a 12-year-old boy with fever, abdominal pain, and diarrhea. METHODS We used pulsed-field gel electrophoresis and analysis of plasmids and beta-lactamases to compare the ceftriaxone-resistant S. enterica serotype typhimurium from the child with four isolates of this strain obtained from cattle during a local outbreak of salmonellosis. RESULTS The ceftriaxone-resistant isolate from the child was indistinguishable from one of the isolates from cattle, which was also resistant to ceftriaxone. Both ceftriaxone-resistant isolates were resistant to 13 antimicrobial agents; all but one of the resistance determinants were on a conjugative plasmid of 160 kb that encoded the functional group 1 beta-lactamase CMY-2. Both ceftriaxone-resistant isolates were closely related to the three other salmonella isolates obtained from cattle, all of which were susceptible to ceftriaxone. CONCLUSIONS This study provides additional evidence that antibiotic-resistant strains of salmonella in the United States evolve primarily in livestock. Resistance to ceftriaxone, the drug of choice for invasive salmonella disease, is a public health concern, especially with respect to children, since fluoroquinolones, which can also be used to treat this disease, are not approved for use in children.

Establishment of a Universal Size Standard Strain for Use with the PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocols: Converting the National Databases to the New Size Standard

ABSTRACT The PulseNet National Database, established by the Centers for Disease Control and Prevention in 1996, consists of pulsed-field gel electrophoresis (PFGE) patterns obtained from isolates of food-borne pathogens (currently Escherichia coli O157:H7, Salmonella, Shigella, and Listeria) and textual information about the isolates. Electronic images and accompanying text are submitted from over 60 U.S. public health and food regulatory agency laboratories. The PFGE patterns are generated according to highly standardized PFGE protocols. Normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. Originally, a well-characterized strain of each organism was chosen as the reference standard for that particular database. The increasing number of databases, difficulty in identifying an organism-specific standard for each database, the increased range of band sizes generated by the use of additional restriction endonucleases, and the maintenance of many different organism-specific strains encouraged us to search for a more versatile and universal DNA size marker. A Salmonella serotype Braenderup strain (H9812) was chosen as the universal size standard. This strain was subjected to rigorous testing in our laboratories to ensure that it met the desired criteria, including coverage of a wide range of DNA fragment sizes, even distribution of bands, and stability of the PFGE pattern. The strategy used to convert and compare data generated by the new and old reference standards is described.

Rapid Pulsed-Field Gel Electrophoresis Protocol for Subtyping of Campylobacter jejuni

ABSTRACT We developed a rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping Campylobacter isolates based on the standardized protocols used by PulseNet laboratories for the subtyping of other food-borne bacterial pathogens. Various combinations of buffers, reagents, reaction conditions (e.g., cell suspension concentration, lysis time, lysis temperature, and restriction enzyme concentration), and electrophoretic parameters were evaluated in an effort to devise a protocol that is simple, rapid, and robust. PFGE analysis of Campylobacter isolates can be completed in 24 to 30 h using this protocol, whereas the most widely used current protocols require 3 to 4 days to complete. Comparison of PFGE patterns obtained in six laboratories showed that subtyping results obtained using this protocol are highly reproducible.

Invasive Escherichia coli are a feature of Crohn's disease.

Crohns disease (CD) and ulcerative colitis (UC) are idiopathic inflammatory conditions of the gut. Our goal was to investigate if invasive Escherichia coli strains were present in patients with inflammatory bowel disease (IBD). Bacterial strains were isolated from biopsy material obtained from normal controls, and patients with a clinical diagnosis of CD and UC. Invasive bacteria were characterized by gentamicin protection assay and biochemical profiling (Api-20E). Strains were characterized by induction of cytokine expression in epithelial and macrophage cell cultures, measurement of epithelial barrier function, and confocal microscopy. Of all invasive bacterial strains in CD 98.9% were identified as E. coli as opposed to 42.1% in UC and 2.1% in normal controls. Epithelial invasion in vitro was significantly higher for CD-associated E. coli (8.4%, ±5.5 of initial inoculum (I/O)) in comparison to UC (2.5%, ±0.4 I/O), but highest for strains from inflamed CD tissue (11.3%, ±4.3 I/O). Both, CD and UC E. coli strains induced high mean TNF-α expression in macrophage cell lines (2604.8 pg/105 cells, ±447.4; 2,402.6 pg/105 cells, ±476.3, respectively), but concentrations were significantly higher for isolates from inflamed CD tissue (3071.3 pg/105 cells, ±226.0). Invasive E. coli from IBD tissue induced similar concentrations of interleukin (IL)-8 in epithelial cell cultures, but strains from inflamed CD tissue induced significantly less epithelial IL-8 (674.1 pg/105 cells, ±58.0 vs 920.5 pg/105 cells, ±94.6). IBD-associated E. coli strains significantly decreased transepithelial resistance, induced disorganization of F-actin and displacement of ZO-1, and E-cadherin from the apical junctional complex (AJC). In comparison to normal controls and UC, E. coli are more prevalent in CD, are highly invasive, and do not encode for known effector proteins. E. coli strains from IBD patients regulate cytokine expression and epithelial barrier function, two pathological features of IBD.

Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States.

Molecular subtyping of Bacillus anthracis played an important role in differentiating and identifying anthrax strains during the 2001 bioterrorism-associated outbreak. Because B. anthracis has a low level of genetic variability, only a few subtyping methods, with varying reliability, exist. We initially used multiple-locus variable-number tandem repeat analysis (MLVA) to subtype 135 B. anthracis isolates associated with the outbreak. All isolates were determined to be of genotype 62, the same as the Ames strain used in laboratories. We sequenced the protective antigen gene (pagA) from 42 representative outbreak isolates and determined they all had a pagA sequence indistinguishable from the Ames strain (PA genotype I). MLVA and pagA sequencing were also used on DNA from clinical specimens, making subtyping B. anthracis possible without an isolate. Use of high-resolution molecular subtyping determined that all outbreak isolates were indistinguishable by the methods used and probably originated from a single source. In addition, subtyping rapidly identified laboratory contaminants and non-outbreak–related isolates.

Salmonella enterica Serotype Typhimurium DT104 Isolated from Humans, United States, 1985, 1990, and 1995

First isolated from an ill person in 1985, multidrug-resistant Salmonella enterica serotype Typhimurium DT104 emerged in the mid-1990s as a strain of Salmonella frequently isolated from humans in the United States. We compared the integron content, plasmid profile, and XbaI pulsed-field gel electrophoresis (PFGE) patterns of multidrug-resistant S. Typhimurium DT104 (MR-DT104) isolated from humans in the United States in 1985, 1990, and 1996. All isolates contained a 60-mDa plasmid and had indistinguishable PFGE and integron profiles, supporting the idea of a clonal relationship between recent and historical isolates. The data suggest that the widespread emergence of MR-DT104 in humans and animals in the 1990s may have been due to the dissemination of a strain already present in the United States rather than the introduction of a new strain.

Novel Virulence Gene and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Multilocus Sequence Typing Scheme for Subtyping of the Major Serovars of Salmonella enterica subsp. enterica

ABSTRACT Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:−, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.

From Pig to Pacifier: Chitterling-Associated Yersiniosis Outbreak among Black Infants

In this case-control study of Yersinia enterocolitica infections among black infants, chitterling preparation was significantly associated with illness (p<0.001). Of 13 samples of chitterlings tested, 2 were positive for Yersinia intermedia and 5 for Salmonella. Decontamination of chitterlings before sale with methods such as irradiation should be strongly considered.

Recent developments and future prospects in subtyping of foodborne bacterial pathogens

Infections caused by foodborne bacterial pathogens continue to be a major public health issue around the world. During the past decade, pulsed-field gel electrophoresis (PFGE) has become the gold standard for molecular subtyping and source tracking of most foodborne bacteria. Owing to problems inherent in PFGE technology, new methods have been developed focusing on DNA sequence-based subtyping. This review discusses the feasibility of using multilocus sequence typing, multiple-locus variable-number tandem repeat analysis, single nucleotide polymorphisms, microarrays, whole genome sequencing and mass spectrometry for subtyping foodborne bacterial pathogens.

Subtyping Salmonella enterica Serovar Enteritidis Isolates from Different Sources by Using Sequence Typing Based on Virulence Genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)

ABSTRACT Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)—CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)—was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.