Peter Gerner-Smidt

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.

Thermal inactivation of Salmonella in peanut butter.

The objective of this study was to determine the rates of thermal inactivation of three Salmonella Tennessee strains in peanut butter associated with an outbreak and to compare them to the rates of inactivation of Salmonella strains of other serotypes (Enteritidis, Typhimurium, and Heidelberg) (SSOS) and of clinical isolates of Salmonella Tennessee from sporadic cases (STSC). Commercial peanut butter was inoculated with Salmonella isolates and heated at 71, 77, 83, and 90 degrees C. The thermal inactivation curves were upwardly concave, indicating rapid death at the beginning (20 min) of heating followed by lower death rates thereafter. The first-order kinetics approach and nonlinear Weibull model were used to fit the inactivation curves and describe the rates of thermal inactivation of Salmonella in peanut butter. The calculated minimum times needed to obtain a 7-log reduction at 90 degrees C for the composited three outbreak-associated strains were significantly greater (P < 0.05) than those of SSOS and STSC. Approximately 120 min were needed to reduce the outbreak strains of Salmonella Tennessee by 7 log, whereas 86 and 55 min were needed for SSOS and STSC, respectively. These results indicate that the outbreak-associated Salmonella strains were more thermotolerant than the other Salmonella strains tested, and this greater thermal resistance was not serotype specific. Thermal treatments of peanut butter at 90 degrees C for less than 30 min are not sufficient to kill large populations (5 log CFU/g) of Salmonella in highly contaminated peanut butter.

Comparative Genomics of Vibrio cholerae from Haiti, Asia, and Africa

A strain from Haiti shares genetic ancestry with those from Asia and Africa.

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.

Genomic Characterization of Listeria monocytogenes Strains Involved in a Multistate Listeriosis Outbreak Associated with Cantaloupe in US

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.

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.

Laboratory-Based Surveillance of Paratyphoid Fever in the United States : Travel and Antimicrobial Resistance

BACKGROUND The incidence of paratyphoid fever, including paratyphoid fever caused by antimicrobial-resistant strains, is increasing globally. However, the epidemiologic and laboratory characteristics of paratyphoid fever in the United States have never been studied. METHODS We attempted to interview all patients who had been infected with laboratory-confirmed Salmonella serotypes Paratyphi A, Paratyphi B, or Paratyphi C in the United States with specimens collected from 1 April 2005 through 31 March 2006. At the Centers for Disease Control and Prevention (CDC), isolates underwent serotype confirmation, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis typing. RESULTS Of 149 patients infected with Salmonella Paratyphi A, we obtained epidemiologic information for 89 (60%); 55 (62%) of 86 were hospitalized. Eighty-five patients (96%) reported having travel internationally, and 80 (90%) had traveled to South Asia. Of the 146 isolates received at the CDC, 127 (87%) were nalidixic acid resistant; nalidixic acid resistance was associated with travel to South Asia (odds ratio, 17.0; 95% confidence interval, 3.8-75.9). All nalidixic acid-resistant isolates showed decreased susceptibility to ciprofloxacin (minimum inhibitory concentration, > or = 0.12 microg/mL). Of 49 patients infected with Salmonella Paratyphi B, only 12 (24%) were confirmed to have Paratyphi B when tested at the CDC. Four (67%) of 6 patients were hospitalized, and 5 (83%) reported travel (4 to the Andean region of South America). One case of Salmonella Paratyphi C infection was reported in a traveler to West Africa with a urinary tract infection. CONCLUSIONS Physicians should be aware of the increasing incidence of infection due to Salmonella Paratyphi A and treatment options given its widespread antimicrobial resistance. A paratyphoid fever vaccine is urgently needed. Continued surveillance for paratyphoid fever will help guide future prevention and treatment recommendations.

Listeria monocytogenes: Maternal-foetal infections in Denmark 1994–2005

Maternal-foetal infection by Listeria monocytogenes is a rare complication in pregnancy. In the period 1994–2005, 37 culture-confirmed cases of maternal-foetal Listeria monocytogenes infections were reported in Denmark. We examined 36 patients’ files in order to evaluate risk factors, clinical and laboratory findings, response to therapy, and outcome for maternal-foetal listeriosis. Patient data and bacteriological findings were divided into 2 groups for comparison: 1 group with children born alive (n=24) and another group with abortion or stillbirth (n=12). 23 of the 36 children survived the acute infection, as did all the mothers. The mothers were generally only mildly affected by the infection. In contrast, among the children born alive, 15 were diagnosed with bacteraemia/septicaemia, 3 children with pneumonia, 3 with neonatal meningitis, and 3 were unaffected. Despite the high frequency of illness only 1 of the live-born children died from the infection and none of the surviving children showed signs of permanent damage at the time they were discharged from hospital. Listeriosis during pregnancy is a serious threat to the unborn child. One-third of culture-confirmed cases of maternal-foetal infections resulted in abortion or stillbirth; however, the prognosis for live-born children is good, even in severely ill newborns.

Molecular surveillance of shiga toxigenic Escherichia coli O157 by PulseNet USA.

PulseNet USA is the national molecular subtyping network system for foodborne disease surveillance. Sixty-four public health and food regulatory laboratories participate in PulseNet USA and routinely perform pulsed-field gel electrophoresis of Shiga toxigenic Escherichia coli isolated from humans, food, water, and the environment on a real-time basis. Clusters of infection are detected in three ways within this system: through rapidly alerting the participants in the electronic communication forum, the PulseNet Web conference; through cluster analysis by the database administrators at the coordinating center at the Centers for Disease Control and Prevention of the patterns uploaded to the central server by the participants; and by matching profiles of strains from nonhuman sources with recent human uploads to the national server. The strengths, limitations, and scope for future improvements of PulseNet are discussed with examples from 2002. In that year, notices of 30 clusters of Shiga toxigenic E. coli O157 infections were posted on the Web conference, 26 of which represented local outbreaks, whereas four were multistate outbreaks. Another 27 clusters were detected by central cluster detection performed at the Centers for Disease Control and Prevention, of which five represented common source outbreaks confirmed after finding an isolate with the outbreak pattern in the implicated food. Ten food isolates submitted without suspicion of an association to human disease matched human isolates in the database, and an epidemiologic link to human cases was established for six of them.

Development and application of MLVA methods as a tool for inter-laboratory surveillance

Multiple-locus variable-number of tandem-repeats analysis (MLVA) has emerged as a valuable method for subtyping bacterial pathogens and has been adopted in many countries as a critical component of their laboratory-based surveillance. Lack of harmonisation and standardisation of the method, however, has made comparison of results generated in different laboratories difficult, if not impossible, and has therefore hampered its use in international surveillance. This paper proposes an international consensus on the development, validation, nomenclature and quality control for MLVA used for molecular surveillance and outbreak detection based on a review of the current state of knowledge.