Patient 2 was a 54-year-old man who was admitted to the same hospital in February 2004 because of sudden fever and confusion. He tested HIV-positive in 1993 (CDC Classification B3), and his CD4+ cell count nadir was 121 cells/mm3. His recent antiretroviral therapy was a combination of stavudine, lamivudine, and nelfinavir. HCV-related liver cirrhosis was diagnosed 3 years before this admission. His condition was routinely followed up in the outpatient unit, and he had been hospitalized 1 month earlier for culture-negative pneumonia in the left lower lobe. On admission, the patient had a high fever (41 C), chills, and abnormal mental status with somnolence. Results of a chest radiograph, nuclear magnetic resonance imaging of the brain, and echocardiogram were normal. HIV viremia level was <50 copies/mL and CD4+ cell count was 204 cells/mm3. Laboratory values were notable for leukocytosis (leukocytes, 14 x 103/mm3), anemia (hemoglobin, 10 g/dL), and thrombocytopenia (platelets, 69 x 103/mm3). Levels of C reactive protein, aminotransferases, blood glucose, creatinine, urea nitrogen, and electrolytes were within the normal range. Liver cirrhosis was classified as Child-Pugh group B, score 7. Urine was negative for opioids and cocaine metabolites. On second day postadmission, multiple blood cultures became positive for Y. pseudotuberculosis, while stool cultures were negative. Intravenous ceftriaxone therapy was begun at admission, with total remission of symptoms within 3 days. He was discharged on day 10 and continued intravenous ceftriaxone therapy for 4 weeks (2 g daily) as an outpatient. At the final clinical observation, 6 months later, the patient was free of clinical symptoms and had no recurrence of Y. pseudotuberculosis infection.
Blood culture isolates from both patients were identified with >99% confidence as Y. pseudotuberculosis by using both the API20E-Rapid test (bioMerieux, Marcy l'Etoile, France) and the BD Phoenix system (Becton Dickinson, Sparks MD, USA). Isolates showed an extended spectrum of antimicrobial susceptibility (sensitive to beta-lactams, monobactams, aminoglycosides, quinolones, and sulfonamides). O-serotyping with commercial antisera (Denka Seiken, Tokyo, Japan) identified both organisms as Y. pseudotuberculosis serotype O1, consistent with the high frequency of this serotype among strains isolated from patients with septicemia in Europe.
The 16S ribosomal DNA (rDNA) sequences were identical in the 2 isolates and optimally matched (>99% identity) the 16S rDNA of the Y. pseudotuberculosis type strain ATCC 29833 (GenBank accession no. AF366375). To differentiate the strains at the genome level, amplified fragment length polymorphism and arbitrarily primed polymerase chain reaction (PCR) were performed. While these methods are highly discriminatory and detect DNA polymorphisms to the strain level, no differences between the 2 clinical isolates were observed (Figure, panels A and B).
To gain insight into the pathogenic potential of the 2 isolates, we assessed the presence of pYV (70-kb virulence plasmid), high pathogenicity island (HPI), and Y. pseudotuberculosis-derived mitogen (YPM) genetic markers by PCR. Both isolates harbored a characteristic repertoire of virulence determinants. Correctly sized PCR products were obtained for inv (chromosome-borne), irp1, irp2, IS100 (HPI-borne), lcrF, and yadA (plasmidborne) genes (Figure, panel C). The identity of PCR products was confirmed by direct DNA sequencing. Interestingly, both isolates were negative for the ypmA (chromosome-borne) gene, consistent with the genetic instability of this marker which is typically absent in strains from Western countries. Reducing PCR stringency for ypmA yielded a 237-bp amplicon, which resulted from mispriming of oligonucleotides on the Y. pseudotuberculosis hmp flavohemoprotein gene (data not shown).
Molecular typing and virulence gene probing indicate that both cases resulted from indistinguishable Y. pseudotuberculosis strains, which raises the suspicion of a common source of infection. However, no other cases of Y. pseudotuberculosis infection were diagnosed in our institute or reported to the National Center for Enteropathogenic Bacteria, Istituto Superiore di Sanita, Rome, from January 2003 to June 2004 (I. Luzzi, pers. comm.). In September 2004, patients were interviewed about their lifestyle, illness, food and fluid consumption, or animal exposure in the 2 weeks preceding hospitalization. Nosocomial infection and direct contact between patients were ruled out, which suggested that infection had been independently acquired in the community. Both patients denied any contact with wild or domestic animals. Patient 1 had not been released from detention in the 6 months preceding hospitalization and regularly consumed collective meals in prison. However, prison infirmary records did not show an increased frequency of abdominal symptoms or fever among  500 inmates from May to July 2003. Patient 2 was a heavy smoker who had been abusing alcohol and illicit drugs until 2001. He lived alone and had a seafood meal 4 days before admission. Thus, although infection may have been acquired from contaminated food or fluid, questions regarding the actual source of bacteria and the extent of exposure remain unanswered.