Frida Stock

Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella pneumoniae with Whole-Genome Sequencing

Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing revealed its origin and probable modes of transmission. A Detective Story Some infections are largely a thing of the past—plague, syphilis. The unfortunate result of these antibiotic-driven successes is the emergence of drug-resistant pathogens. And, ironically enough, hospitals are at the center of the problem. An example of this occurred in 2011 at the Clinical Center of the U.S. National Institutes of Health (NIH), in which an outbreak of drug-resistant Klebsiella pneumoniae infected 18 patients, causing the death of 6 of them. Using a combination of whole-genome sequencing and patient tracking, Snitkin and his colleagues examined how the bacteria was spreading through the hospital. The results outline a complicated path of transmission within the hospital that defied standard containment methods, yielding lessons for the future. A patient known to be infected with a drug-resistant form of K. pneumoniae was admitted to the NIH Clinical Center on 13 June 2011. Enhanced isolation procedures were immediately implemented, and no spread of the bacteria was seen for the month she was in the hospital. Although all seemed well, a few weeks later on August 5th, a second infected patient was discovered, followed by a series of other patients with infection or colonization—about 1 a week to a total of 18 by the end of 2011. Six people ultimately died as a result of the bacteria. The outbreak was finally contained by rigorous control procedures. A careful survey of the bed locations of each patient did not shed much light on how the bacteria traveled on its deadly path: The first patient did not even come into contact with any of the others. So the authors performed whole-genome sequencing on all of the bacteria that were found, determining the most likely evolutionary relationships among them by comparing the variations at single nucleotides that arise as bacteria grow. Combining this evolutionary information with the physical tracking of the patients pointed to the most likely transmission scenario. The authors concluded that all of the K. pneumoniae cases likely originated with the index patient, from at least two different sites on her body, rather than by independently introduced bacteria. There were at least three different initial transmission events. Particularly disturbing was the fact that one of the infections could be linked to contamination of a ventilator that had been cleaned by thorough methods. Sophisticated deployment of whole-genome sequencing revealed the weaknesses in this medical who-done-it, informing improvements in hospital preventive measures. If applied rapidly, such analysis can even expose the causes of nosocomial infections in real time. The Gram-negative bacteria Klebsiella pneumoniae is a major cause of nosocomial infections, primarily among immunocompromised patients. The emergence of strains resistant to carbapenems has left few treatment options, making infection containment critical. In 2011, the U.S. National Institutes of Health Clinical Center experienced an outbreak of carbapenem-resistant K. pneumoniae that affected 18 patients, 11 of whom died. Whole-genome sequencing was performed on K. pneumoniae isolates to gain insight into why the outbreak progressed despite early implementation of infection control procedures. Integrated genomic and epidemiological analysis traced the outbreak to three independent transmissions from a single patient who was discharged 3 weeks before the next case became clinically apparent. Additional genomic comparisons provided evidence for unexpected transmission routes, with subsequent mining of epidemiological data pointing to possible explanations for these transmissions. Our analysis demonstrates that integration of genomic and epidemiological data can yield actionable insights and facilitate the control of nosocomial transmission.

Phase I Study of the Intravenous Administration of Attenuated Salmonella typhimurium to Patients With Metastatic Melanoma

PURPOSE A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice. These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer. PATIENTS AND METHODS In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 10(6) to 10(9) cfu/m(2) of VNP20009. Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects. RESULTS The maximum-tolerated dose was 3 x 10(8) cfu/m(2). Dose-limiting toxicity was observed in patients receiving 1 x 10(9) cfu/m(2), which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 x 10(9) cfu/m(2) and in one patient receiving 3 x 10(8) cfu/m(2). None of the patients experienced objective tumor regression, including those patients with colonized tumors. CONCLUSION The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed. No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.

Cohort Study of Molecular Identification and Typing of Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii

ABSTRACT Mycobacterium abscessus is the most common cause of rapidly growing mycobacterial chronic lung disease. Recently, two new M. abscessus-related species, M. massiliense and M. bolletii, have been described. Health care-associated outbreaks have recently been investigated by the use of molecular identification and typing tools; however, very little is known about the natural epidemiology and pathogenicity of M. massiliense or M. bolletii outside of outbreak situations. The differentiation of these two species from M. abscessus is difficult and relies on the sequencing of one or more housekeeping genes. We performed extensive molecular identification and typing of 42 clinical isolates of M. abscessus, M. massiliense, and M. bolletii from patients monitored at the NIH between 1999 and 2007. The corresponding clinical data were also examined. Partial sequencing of rpoB, hsp65, and secA led to the unambiguous identification of 26 M. abscessus isolates, 7 M. massiliense isolates, and 2 M. bolletii isolates. The identification results for seven other isolates were ambiguous and warranted further sequencing and an integrated phylogenetic analysis. Strain relatedness was assessed by repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE), which showed the characteristic clonal groups for each species. Five isolates with ambiguous species identities as M. abscessus-M. massiliense by rpoB, hsp65, and secA sequencing clustered as a distinct group by rep-PCR and PFGE together with the M. massiliense type strain. Overall, the clinical manifestations of disease caused by each species were similar. In summary, a multilocus sequencing approach (not just rpoB partial sequencing) is required for division of M. abscessus and closely related species. Molecular typing complements sequence-based identification and provides information on prevalent clones with possible relevant clinical aspects.

Genome-wide recombination drives diversification of epidemic strains of Acinetobacter baumannii

Acinetobacter baumannii is an emerging human pathogen and a significant cause of nosocomial infections among hospital patients worldwide. The enormous increase in multidrug resistance among hospital isolates and the recent emergence of pan-drug–resistant strains underscores the urgency to understand how A. baumannii evolves in hospital environments. To this end, we undertook a genomic study of a polyclonal outbreak of multidrug-resistant A. baumannii at the research-based National Institutes of Health Clinical Center. Comparing the complete genome sequences of the three dominant outbreak strain types enabled us to conclude that, despite all belonging to the same epidemic lineage, the three strains diverged before their arrival at the National Institutes of Health. The simultaneous presence of three divergent strains from this lineage supports its increasing prevalence in international hospitals and suggests an ongoing adaptation to the hospital environment. Further genomic comparisons uncovered that much of the diversification that occurred since the divergence of the three outbreak strains was mediated by homologous recombination across 20% of their genomes. Inspection of recombinant regions revealed that several regions were associated with either the loss or swapping out of genes encoding proteins that are exposed to the cell surface or that synthesize cell-surface molecules. Extending our analysis to a larger set of international clinical isolates revealed a previously unappreciated ability of A. baumannii to vary surface molecules through horizontal gene transfer, with subsequent intraspecies dissemination by homologous recombination. These findings have immediate implications in surveillance, prevention, and treatment of A. baumannii infections.

A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91phox −/− (X-linked) and p47 phox −/− (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patients original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Kochs postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis.

Resistance to Multiple Fluoroquinolones in a Clinical Isolate of Streptococcus pyogenes: Identification of gyrA and parC and Specification of Point Mutations Associated with Resistance

ABSTRACT A strain of Streptococcus pyogenes resistant to multiple fluoroquinolones was isolated from the blood of an immunocompromised patient. Resistance to fluoroquinolones in S. pyogenes has not been previously studied. Compared to 10 sensitive strains of S. pyogenes, the fluoroquinolone-resistant clinical isolate of S. pyogenespresented point mutations in gyrA, predicting that serine-81 was changed to phenylalanine and that methionine-99 was changed to leucine, and in parC, predicting that serine-79 was changed to tyrosine. The mechanism of fluoroquinolone resistance in this isolate of S. pyogenes appears to be analogous to previously reported mechanisms for Streptococcus pneumoniae.

Mechanisms of Resistance to Daptomycin in Enterococcus faecium

ABSTRACT In this study, we investigated the clonal emergence of daptomycin-resistant Enterococcus faecium strains isolated from a patient with leukocyte adhesion deficiency syndrome. The resistance mechanism in these strains is independent of either equivalent point mutations previously described for Staphylococcus aureus or daptomycin inactivation mechanisms identified in soil bacteria.

Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. carinii

ABSTRACT A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r = 0.99) over 6 log values for standards containing ≥5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.

The use of oral washes to diagnose Pneumocystis carinii pneumonia: A blinded prospective study using a polymerase chain reaction-based detection system

Pneumocystis carinii pneumonia (PCP) can be diagnosed by direct microscopic examination of induced sputum or by bronchoalveolar lavage (BAL). However, many institutions have little diagnostic success with induced sputum, and BAL is invasive and expensive. This prospective, blinded study assessed oral washes as a more convenient specimen than either sputum or BAL fluid and used a dissociation-enhanced lanthanide fluoroimmunoassay time-resolved fluorescent hybridization polymerase chain reaction (PCR) detection system that is feasible for clinical laboratories. The study assessed 175 oral washes, each paired with either an induced sputum that was positive for Pneumocystis or a BAL sample. The PCR test based on the Pneumocystis major surface glycoprotein primers had a sensitivity of 91% and a specificity of 94%, compared with a test based on mitochondrial large subunit rRNA primers, which had a sensitivity of 75% and a specificity of 96%. These results suggest that oral washes can provide a useful sample for diagnosis of PCP when a sensitive PCR detection system is used.

Reinfection, Rather than Persistent Infection, in Patients with Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is characterized by severe recurrent infections with Staphylococcus aureus, certain gram-negative rods, Nocardia species, and fungi. When infections with the same species recur, they may represent relapses or new infections. We collected organisms from infections that occurred between 1992 and 2000 in patients with CGD and determined the biochemical phenotypes, in vitro antibiotic susceptibility patterns, and pulsed-field gel electrophoresis (PFGE) patterns of the organisms causing the initial and recurrent infections. Recurrence of infection with Burkholderia cepacia or Serratia marcescens was caused by a new strain in 9 of 10 cases (P=.001). Recurrent S. aureus infections were caused by new strains in 7 of 8 cases (P=.006). In patients with CGD, recurrence of infection with the same bacterial species after appropriate antibiotic therapy usually represents new infection.