- Volume 57, Issue 10, 2008
Volume 57, Issue 10, 2008
- Pathogenicity And Virulence
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An intracellularly inducible gene involved in virulence and polyphosphate production in Francisella
More LessFrancisella tularensis is an intracellular pathogen capable of multiplying to high levels in macrophages. By protein analysis, only a few proteins have been shown previously to be expressed at high levels in macrophages relative to bacteria grown in culture media. To identify additional genes that show increased expression during intracellular growth, we developed a plasmid for use in Francisella based on the induction of expression of green fluorescent protein. Clones of F. tularensis subsp. novicida were identified that were fluorescent only intracellularly and not when grown in vitro. Sequencing identified a range of genes comprising some such as dnaK that are already known to be expressed intracellularly and some novel targets. One of these newly identified regulated genes, FTN1472/FTT1564, was selected for further study. Isogenic mutants were generated in F. tularensis subsp. novicida and subsp. tularensis by allelic replacement. Inactivation of the gene resulted in abolition of polyphosphate production by F. novicida, strongly supporting the bioinformatic analysis, which had suggested that the gene may encode a polyphosphate kinase. The mutants exhibited defects for intracellular growth in macrophages and were attenuated in mice, indicating a key role for the putative polyphosphate kinase in the virulence of Francisella.
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- Host Response
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Dichotomous metabolism of Enterococcus faecalis induced by haematin starvation modulates colonic gene expression
Enterococcus faecalis is an intestinal commensal that cannot synthesize porphyrins and only expresses a functional respiratory chain when provided with exogenous haematin. In the absence of haematin, E. faecalis reverts to fermentative metabolism and produces extracellular superoxide that can damage epithelial-cell DNA. The acute response of the colonic mucosa to haematin-starved E. faecalis was identified by gene array. E. faecalis was inoculated into murine colons using a surgical ligation model that preserved tissue architecture and homeostasis. The mucosa was exposed to haematin-starved E. faecalis and compared with a control consisting of the same strain grown with haematin. At 1 h post-inoculation, 6 mucosal genes were differentially regulated and this increased to 42 genes at 6 h. At 6 h, a highly significant biological interaction network was identified with functions that included nuclear factor-κB (NF-κB) signalling, apoptosis and cell-cycle regulation. Colon biopsies showed no histological abnormalities by haematoxylin and eosin staining. Immunohistochemical staining, however, detected NF-κB activation in tissue macrophages using antibodies to the nuclear localization sequence for p65 and the F4/80 marker for murine macrophages. Similarly, haematin-starved E. faecalis strongly activated NF-κB in murine macrophages in vitro. Furthermore, primary and transformed colonic epithelial cells activated the G2/M checkpoint in vitro following exposure to haematin-starved E. faecalis. Modulation of this cell-cycle checkpoint was due to extracellular superoxide produced as a result of the respiratory block in haematin-starved E. faecalis. These results demonstrate that the uniquely dichotomous metabolism of E. faecalis can significantly modulate gene expression in the colonic mucosa for pathways associated with inflammation, apoptosis and cell-cycle regulation.
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- Diagnostics, Typing And Identification
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Molecular detection methods and serotyping performed directly on clinical samples improve diagnostic sensitivity and reveal increased incidence of invasive disease by Streptococcus pneumoniae in Italian children
The aims of this study were to evaluate the incidence of invasive pneumococcal disease (IPD) in Italian children and perform serotyping by PCR-based assays directly on clinical samples. A 1-year paediatric (0–14 years) population-based surveillance study was designed to evaluate the incidence of IPD in the province of Florence, Italy, by cultural and molecular methods. Among 92 children (80 with pneumonia, 8 with meningitis/sepsis, 4 with arthritis), 4 cases of IPD were diagnosed both by culture and real-time PCR and 18 cases exclusively by molecular methods. The sensitivity of molecular methods was significantly higher than that of cultural methods (Cohen’s κ 0.41; McNemar P=0.000008). The incidence of IPD in children below 2 years of age was 11.5/100 000 and 51.8/100 000 by cultural and molecular methods, respectively. Pneumococcal serotyping by multiplex sequential PCR was obtained in 19/22 samples. Real-time PCR and multiplex sequential PCR can be used directly on biological samples, improving the ability to diagnose IPD. The incidence of IPD appears 5–10 times higher by PCR than by cultural methods.
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A novel method for simple detection of mutations conferring drug resistance in Mycobacterium leprae, based on a DNA microarray, and its applicability in developing countries
A simple method to detect mutations in the genome of Mycobacterium leprae that confer resistance to key drugs for leprosy was exploited on the basis of a reverse hybridization system. A series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB and gyrA genes for dapsone, rifampicin and ofloxacin resistance, respectively, were selected and fixed on a glass slide as capture probes, to develop a DNA microarray termed the leprosy drug susceptibility-DNA microarray (LDS-DA). Mutations in clinical isolates of M. leprae were successfully identified by the LDS-DA. Feasibility studies were conducted to evaluate the performance of the LDS-DA in two developing countries, Myanmar and the Philippines. The high concordance of the results obtained by this method with the results of nucleotide sequencing strongly supports the applicability of the LDS-DA as a drug susceptibility test in place of sequencing, a time-consuming and costly procedure. This is a rapid and simple method for the simultaneous susceptibility testing of three front-line drugs for leprosy, and solves the problems of previously reported methods.
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An evaluation of the recovery of mycobacteria from urine specimens using the automated Mycobacteria Growth Indicator Tube system (BACTEC MGIT 960)
More LessThe Mycobacteria Growth Indicator Tube (MGIT 960) system was evaluated against Lowenstein–Jensen (LJ) medium and the BACTEC 460 TB system for the recovery of mycobacteria from 1393 consecutive urine specimens. The MGIT had a sensitivity of 91.3 % [95 % confidence interval (CI), 83.2–99.4] when the combination of BACTEC 460 and LJ medium was used as the reference method. The mean time for positivity for MGIT and BACTEC 460 was 19.3 days and 20 days, respectively, while that for LJ medium was 35 days.The incidence of contamination was highest for LJ medium (n=148), followed by MGIT 960 (n=81), and BACTEC 460 had the lowest incidence of contamination (n=50). In conclusion, the isolation of mycobacteria from urine specimens by the MGIT 960 is comparable to that of the BACTEC 460 TB system and solid media.
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Development of a PCR assay for the identification of Salmonella enterica serovar Brandenburg
More LessCurrently, Salmonella enterica serovar Brandenburg is identified serologically on the basis of two surface antigens, somatic (O) polysaccharide and flagellar (H) proteins. This procedure is time-consuming and requires expensive typing reagents. To overcome these problems, a PCR method was developed and validated for the identification of S. Brandenburg. Portions of the invA, rfbJ(B), fliC and fljB genes were targeted for amplification using four pairs of oligonucleotide primers. To validate the assay, genomic DNA from an array of 72 Salmonella strains representing 28 serotypes and 5 non-Salmonella strains from 4 different genera was subjected to PCR. The four targeted genes were correctly amplified only from S. Brandenburg. These results indicate that this PCR assay is a simple, rapid, reliable and reproducible method for the identification of S. Brandenburg that will aid in surveillance, prevention and control of this pathogen.
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Screening and confirmation of human immunodeficiency virus type 1 infection solely by detection of RNA
Diagnosis of human immunodeficiency virus (HIV) infection by antibody-based testing allows for some recently infected individuals to be falsely assessed as non-infected. Since such individuals often have high viral loads and are capable of transmitting HIV, it is an imperative public health need to identify these individuals. We investigated the feasibility and capability of a diagnostic algorithm which included screening and confirmation of HIV infection using only nucleic-acid-based tests. This investigation involved screening 1361 prospectively collected specimens using antibody-based methods in parallel to simultaneously testing the same specimens by a qualitative HIV RNA detection method (APTIMA HIV-1). Specimens that were positive by antibody screening were confirmed by either immunofluorescent assay or Western blotting, while specimens positive by RNA screening were confirmed by real-time RT-PCR. In the course of the study, 27 specimens were found to contain either HIV antibody or HIV RNA. Twenty-six of the 27 specimens were HIV RNA positive, while 23 of the 27 specimens were antibody positive. One specimen was found which possessed HIV antibody but was assessed as negative by the HIV RNA screening test. Four specimens were found to contain detectable HIV RNA but were negative by the antibody screening test. Three of these four patients were negative at point-of-care by rapid test, while one was negative by enzyme immunoassay. These data indicate that screening and confirmation of HIV infection by RNA methods alone, if affordable, may constitute an effective alternative HIV diagnostic algorithm in certain settings.
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- Epidemiology
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An unexpectedly high prevalence of colonization with the intestinal spirochaete Brachyspira aalborgi amongst residents of the Indonesian island of Bali
More LessPCR assays designed to amplify DNA from the anaerobic intestinal spirochaete Brachyspira aalborgi were conducted on DNA extracted from 938 faecal samples from 469 residents on the Indonesian island of Bali. The individuals tested were sampled twice in one year and were from four rural villages, one peri-urban centre and the capital city, Denpasar. Overall, an unexpectedly high prevalence of colonization (24.7 %) was found, with prevalence rates at different locations varying from a low of 15.6 % at one village to 41.5 % in the peri-urban centre. Comparison of prevalence rates at the two sampling times suggested that, in many individuals, colonization was likely to be prolonged (>3 months) and/or that reinfection was occurring frequently in these people. Analysis of a questionnaire administered to the individuals who were sampled identified specific risk factors for colonization as location, co-colonization with the related intestinal spirochaete Brachyspira pilosicoli and use of drinking water obtained from wells rather than from taps. No specific associations with clinical symptoms were identified.
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Superantigen genes in group A streptococcal isolates and their relationship with emm types
Superantigens are important virulence factors in the pathogenesis of invasive disease caused by group A streptococcus (GAS). There has been a recent re-emergence of this disease worldwide. A number of novel superantigens have been described recently. This study investigated 107 isolates of GAS for possession of each of the 11 currently known superantigen genes to determine the prevalence, co-occurrence and genetic restriction amongst different emm types of GAS. The results were compared with those in previously published studies. Superantigen genes were not randomly distributed amongst GAS isolates. Certain combinations of superantigen genes were more common and the majority of emm types showed restricted superantigen profiles. This is the first prevalence study of GAS isolates to include the complete range of known superantigen genes and their restriction amongst emm types. This study contributes to the understanding of the relationship between superantigen genes and emm types, and highlights the importance of comprehensive studies in different populations.
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Rising prevalence of enteric fever due to multidrug-resistant Salmonella: an epidemiological study
More LessA prospective study of the prevalent aetiology of enteric fever was undertaken at a tertiary care hospital in North India at intervals of every 3 years. Salmonella spp. were isolated from 174 (7 %) patients. Amongst these, 140 (80 %) patients were infected by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) and 16 (9 %) by S. enterica serovar Paratyphi A; the remaining 11 % were infected by other S. enterica serogroups, Typhimurium, Paratyphi C and Senftenberg, and other group E salmonella. A significantly greater number of S. Typhi were isolated in the summer and monsoon months. Multidrug resistance (resistance to chloramphenicol, ampicillin and co-trimoxazole) sequentially increased from 34 % in 1999 to 66 % in 2005. Increasing resistance was also noticed to the other antibiotics, especially to the cephalosporins. Moreover 8 % of the S. Typhi isolates were found to be presumptive extended spectrum β-lactamase producers. There was a gradual development of resistance to fluoroquinolones over the 7 years. No resistance was observed to fluoroquinolones in 1999, while in 2005 4.4 % resistance was observed to sparfloxacin, 8.8 % resistance to ofloxacin and a high resistance, 13 %, to ciprofloxacin. This is an alarming development and it is of paramount importance to limit unnecessary use of fluoroquinolones and third generation cephalosporins so that their efficacy against salmonella is not jeopardized further.
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Molecular epidemiology and antimicrobial susceptibilities of 273 exfoliative toxin-encoding-gene-positive Staphylococcus aureus isolates from patients with impetigo in Japan
The molecular epidemiology and antimicrobial susceptibilities of 273 Staphylococcus aureus isolates positive for the exfoliative toxin-encoding gene obtained from patients with impetigo in Japan in 2006 were studied. The mecA gene was detected in 74 meticillin-resistant S. aureus (MRSA) and 23 meticillin-susceptible S. aureus (MSSA) isolates. All isolates with the staphylococcal cassette chromosome (SCC) mec were classified into type IV (92.8 %, 90/97) or V (7.2 %, 7/97). The ET-encoding gene etb was found primarily in strains with mecA (87.7 %, 71/81), whilst eta (86.6 %, 161/186) was detected mainly in strains without mecA. The chromosomal enterotoxin-encoding gene cluster egc was found in 83.0 % of strains with eta, whilst no enterotoxin-encoding gene was detected in strains with only etb. PFGE showed that each strain carrying eta, etb and etd could be classified into distinct groups. The susceptibility profiles of MRSA to antimicrobial agents excluding β-lactams were similar to those of MSSA. Gentamicin- and clarithromycin-resistant strains were frequently found for both MRSA and MSSA. The aminoglycoside-resistance gene aacA–aphD was detected in 97.3 % of MRSA and 85.4 % of MSSA. Additionally, the macrolide-resistance gene ermA or ermC was detected in 67.6 % of MRSA and 71.4 % of MSSA. Therefore, these results suggest that SCCmec types IV or V have spread, particularly in MSSA carrying etb in the community.
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- Clinical Microbiology And Virology
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Current epidemiology of intracranial abscesses: a prospective 5 year study
More LessIntracranial abscesses remain a significant health-care problem in developing countries. In view of this, we undertook a comprehensive study to determine the demographics and bacteriological spectrum of brain abscesses in our hospital. Bacteriological profiles and antibiograms were studied by conventional microbiological methods. Seventy-five patients were admitted with brain abscesses over a 5 year period (2001–2005). There was 9.5 % mortality in patients included in this study. The most important factors influencing mortality from intracranial abscess were the age and neurological condition of the patient at the time of admission. Brain abscess could develop at any age but there was a preponderance of males over females. Chronic suppurative otitis media was the most common predisposing factor for temporal lobe infections. Forty-one (54.70 %) abscesses were found to be due to pyogenic organisms, 4 % due to Mycobacterium tuberculosis and 1.3 % were due to Cladophialophora bantiana. The majority of microbial isolates were sensitive to the therapeutic regime adopted in our neurosurgery unit (cefotaxime, gentamicin and metronidazole). Chloramphenicol is another antibiotic with in vitro activity against the isolates.
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Comparison of serum and whole-blood specimens for the detection of Candida DNA in critically ill, non-neutropenic patients
In contrast to the multitude of studies on fungal PCR assay methods, little work has been reported evaluating Candida PCR performance when using whole blood compared with serum in candidaemic patients. Here, a comparison of the performance of whole-blood and serum specimens using a set of real-time PCR Candida species assays is described. Specimens were collected prospectively from non-neutropenic adults who were recruited to a diagnostic clinical trial, the primary purpose of which was to verify the performance of the assays using serum; in all, 104 participants also had whole-blood specimens submitted for analysis in addition to the serum specimen. Of these participants, 10 had laboratory-confirmed candidaemia and 94 were categorized as being ‘unlikely’ to have invasive Candida infection. PCR results from the whole-blood specimens are presented here and compared with the results from serum specimens in this subgroup among whom both specimen types were obtained contemporaneously. All participants with candidaemia were PCR-positive from serum samples; however, only seven were PCR-positive from whole blood. All specimens from patients in the ‘unlikely’ category were PCR-negative in both types of specimen. Moreover, DNA extraction from serum required 1 h; extraction from whole blood required approximately 3 h. These data tentatively suggest that, overall, serum is an appropriate specimen for Candida PCR for detection of candidaemia in non-neutropenic adults.
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Plasmid-borne armA methylase gene, together with bla CTX-M-15 and bla TEM-1, in a Klebsiella oxytoca isolate from China
More LessAn armA-producing Klebsiella oxytoca isolate, strain 157, was detected after screening of 447 extended-spectrum β-lactamase-producing Enterobacteriaceae isolates in China. K. oxytoca 157 was resistant to aminoglycosides, ciprofloxacin and most β-lactams. Resistance to aminoglycosides and β-lactams could be transferred to recipient Escherichia coli by conjugation. armA, bla CTX-M-15 and bla TEM-1 genes were detected in K. oxytoca 157 and transconjugant E. coli strain 600(pEC157). Mutation of aa 87 in GyrA was found in K. oxytoca 157. A plasmid of ∼55 kb was extracted from K. oxytoca 157(pKO157) and E. coli 600(pEC157). Southern blot hybridization confirmed that the armA, bla CTX-M-15 and bla TEM-1 genes were all located on this conjugative plasmid (pEC157). PCR mapping was also performed to investigate the genetic environment of armA. The armA gene was found to be flanked by the same putative transposable elements as reported previously in E. coli, Klebsiella pneumoniae and Citrobacter freundii isolates from different countries.
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- Oral Microbiology
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Phenotypic evaluation of the effect of anaerobiosis on some virulence attributes of Candida albicans
The current assumption that Candida albicans is a facultatively anaerobic organism has been widely accepted since its recovery from anoxic sites became common. However, the link between anaerobiosis and virulence remains uncertain. This study investigated the differential cell-surface hydrophobicity (CSH) using a hydrocarbon/water partition technique and analysed the differential secretion rates of secretory aspartyl proteases (Saps), esterase, chondroitinase and haemolysins of C. albicans strains recovered from periodontal pockets and non-periodontium-related intra-oral sites. For the enzymic tests, all strains from both sets were grown under aerobic and anaerobic conditions and the harvested cells were inoculated onto suitable normal or pre-reduced culture media in the presence or absence of molecular oxygen, respectively. The results showed that no variations were perceptible for CSH and chondroitinase (P >0.05). The secretion rates of esterase and haemolysins strongly decreased in an anoxic environment (P <0.0001). However, a consistent increment (P <0.0001) in Sap secretion was detected when cultures were grown under anaerobic conditions. Based on these results, it is suggested that the oxygen concentration in the atmosphere surrounding cells exerts a variable influence on the virulence attributes of C. albicans.
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- Models Of Infection
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Detection of Haemophilus influenzae type B DNA in a murine pneumonia model by in situ PCR
More LessThis study estimated the value of in situ PCR (ISPCR) in the detection of Haemophilus influenzae type b (Hib) DNA in paraffin-embedded lung tissues of a murine pneumonia model. ICR mice were infected with Hib solution intranasally. In study group A (n=20), physiological changes and the number of deaths were recorded for 7 consecutive days after infection. In study group B (n=10), blood samples and lung tissues were obtained from the infected mice on the brink of death. In both groups, portions of the lung tissue were cultured for Hib, while other portions were submitted for histopathological studies. Conventional PCR, PCR followed by Southern blotting and ISPCR were performed to detect Hib in paraffin-embedded lung tissues. In control group A, six mice were inoculated intranasally with the same concentration of heat-inactivated Hib solution. In control group B, six healthy mice served as a blank control. Both control groups were managed using the same methods as those used in the study groups. The white blood cell count of the mice in the study group increased (F=3.295, P<0.01), with a high neutrophil count (F=0.127, P<0.05). In the histopathological study, various stages of pneumonia were found in the lung tissues of the infected mice examined by microscope; 80 % of the mice had moderate or severe pneumonia. Cultures of lung tissues in the study groups were all positive for Hib, while no bacteria were found in the control groups. Hib was detected in only 4 of 30 samples (13.3 %) of the study groups using conventional PCR, but in all 30 samples (100 %) using both Southern blotting and ISPCR. All three methods did not detect Hib in the control groups. Because of its sensitivity and specificity and its ability to locate the micro-organism, ISPCR can be considered suitable for the detection of Hib in paraffin-embedded lung tissues.
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- Case Reports
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Coccidioidal pericarditis: a rapid presumptive diagnosis by an in-house antigen confirmed by mycological and molecular methods
Coccidioidal pericarditis is a condition found in approximately 1–5 % of patients infected by Coccidioides species. It is associated with widely diverse clinical symptoms. This paper reports a case of coccidioidal pericarditis diagnosed by an in-house Coccidioides posadasii antigen and confirmed with mycological and molecular methods. From February to September 2005, the patient suffered from fever, weight loss, a non-productive cough, thoracic pain and tachycardia. He received a positive diagnosis of coccidioidal pericarditis only in October 2005. The macromorphological examination of the culture showed a whitish felt-like colony, which became brownish with age. Preparations in lactophenol cotton blue stain showed hyaline septate hyphae with fragmentation and thin arthroconidia-like structures. Pericardial fluid and sera samples were positive for Coccidioides antibodies by immunodiffusion and ELISA with a C. posadasii in-house antigen preparation. The C. posadasii identification was confirmed by nested PCR of the antigen 2/proline-rich antigen (Ag2/PRA) encoding gene.
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Successful treatment of Chromobacterium violaceum sepsis in South Africa
More LessChromobacterium violaceum sepsis is extremely rare and usually fatal. A very few cases of C. violaceum infection have been reported from Africa, but never from South Africa. As far as could be ascertained, this infection has never been reported in a patient with leukaemia. We describe what we believe to be the first such case of C. violaceum sepsis, in a 16-year-old female patient with acute biphenotypic leukaemia, which developed during the neutropenic phase after intensive chemotherapy. The infection was due to a non-pigmented strain of C. violaceum and was associated with a co-infection with Candida parapsilosis; both were successfully treated using broad-spectrum antibiotics, antifungals and removal of a Hickman line.
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Two fatal cases of psittacosis caused by Chlamydophila psittaci
More LessTwo fatal cases of psittacosis are described in two poultry-processing-plant employees presenting with pneumonia and respiratory failure. Diagnosis was confirmed by serological and PCR methods. Psittacosis due to Chlamydophila psittaci infection usually has a good recovery rate, although diagnostic delay and mistreatment can lead to severe complications and even death. This report emphasizes the need for rapid differential diagnosis and management of suspected cases of atypical pneumonia to prevent fatal outcomes.
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Endocarditis due to Corynebacterium amycolatum
More LessCorynebacterium amycolatum, a normal inhabitant of human skin, is a Gram-positive, non-spore-forming, mycolic acid-free, aerobic or facultative anaerobic bacillus. Since its description in 1988, it has only rarely been associated with infective endocarditis. This paper describes a case of infective endocarditis successfully treated by combination therapy with daptomycin and rifampicin. To the best of our knowledge, this is the first case report of C. amycolatum endocarditis from the USA successfully treated with these agents.
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Neonatal sepsis caused by a CTX-M-32-producing Escherichia coli isolate
We describe what we believe to be the first case of neonatal sepsis caused by CTX-M-producing Escherichia coli, in a low-weight preterm infant, born to a colonized mother who had received antibiotic treatment antepartum. Increased dissemination of extended-spectrum β-lactamase-producing E. coli in the community should be borne in mind for empirical therapy of sepsis in high-risk newborns.
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- Correspondence
Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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