Experience with fosfomycin for treatment of urinary tract infections due to multidrug-resistant organisms.

Fosfomycin has shown promising in vitro activity against multidrug-resistant (MDR) urinary pathogens; however, clinical data are lacking. We conducted a retrospective chart review to describe the microbiological and clinical outcomes of urinary tract infections (UTIs) with MDR pathogens treated with fosfomycin tromethamine. Charts for 41 hospitalized patients with a urine culture for an MDR pathogen who received fosfomycin tromethamine from 2006 to 2010 were reviewed. Forty-one patients had 44 urinary pathogens, including 13 carbapenem-resistant Klebsiella pneumoniae (CR-Kp), 8 Pseudomonas aeruginosa, and 7 vancomycin-resistant Enterococcus faecium (VRE) isolates, 7 extended-spectrum beta-lactamase (ESBL) producers, and 9 others. In vitro fosfomycin susceptibility was 86% (median MIC, 16 µg/ml; range, 0.25 to 1,024 µg/ml). Patients received an average of 2.9 fosfomycin doses per treatment course. The overall microbiological cure was 59%; failure was due to either relapse (24%) or reinfection UTI (17%). Microbiological cure rates by pathogen were 46% for CR-Kp, 38% for P. aeruginosa, 71% for VRE, 57% for ESBL producers, and 100% for others. Microbiological cure (n = 24) was compared to microbiological failure (n = 17). There were significantly more solid organ transplant recipients in the microbiological failure group (59% versus 21%; P = 0.02). None of the patients in the microbiological cure group had a ureteral stent, compared to 24% of patients within the microbiological failure group (P = 0.02). Fosfomycin demonstrated in vitro activity against UTIs due to MDR pathogens. For CR-KP, there was a divergence between in vitro susceptibility (92%) and microbiological cure (46%). Multiple confounding factors may have contributed to microbiological failures, and further data regarding the use of fosfomycin for UTIs due to MDR pathogens are needed.

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay.

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.

Treatment and outcomes in carbapenem-resistant Klebsiella pneumoniae bloodstream infections.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is an emerging multidrug-resistant nosocomial pathogen. This is a retrospective chart review describing the outcomes and treatment of 60 cases of CR-Kp bloodstream infections. All CR-Kp isolated from blood cultures were identified retrospectively from the microbiology laboratory from January 2007 to May 2009. Clinical information was collected from the electronic medical record. Patients with 14-day hospital mortality were compared to those who survived 14 days. The all-cause in-hospital and 14-day mortality for all 60 CR-Kp bloodstream infections were 58.3% and 41.7%, respectively. In this collection, 98% of tested isolates were susceptible in vitro to tigecycline compared to 86% to colistimethate, 45% to amikacin, and 22% to gentamicin. Nine patients died before cultures were finalized and received no therapy active against CR-Kp. In the remaining 51 patients, those who survived to day 14 (n = 35) were compared to nonsurvivors at day 14 (n=16). These patients were characterized by both chronic disease and acute illness. The 90-day readmission rate for hospital survivors was 72%. Time to active therapy was not significantly different between survivors and nonsurvivors, and hospital mortality was also similar regardless of therapy chosen. Pitt bacteremia score was the only significant factor associated with mortality in Cox regression analysis. In summary, CR-Kp bloodstream infections occur in patients who are chronically and acutely ill. They are associated with high 14-day mortality and poor outcomes regardless of tigecycline or other treatment regimens selected.

False-positive cryptococcal antigen test associated with use of BBL Port-a-Cul transport vials.

A total of 52 residual CSF and serum specimens, which were originally negative with the Cryptococcal Antigen Latex Agglutination System (CALAS), were shown to become falsely positive after placement in BBL Port-A-Cul anaerobic transport vials. This transport device, although excellent for specimen transportation for subsequent culture, should not be used if cryptococcal antigen testing is needed.

Alternative use for spectra MRSA chromogenic agar in detection of methicillin-resistant Staphylococcus aureus from positive blood cultures.

Spectra MRSA agar (Remel, Lenexa, KS), a novel chromogenic medium originally developed to detect methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs, was evaluated in this multicenter study for the detection of MRSA from positive blood cultures exhibiting Gram-positive cocci upon initial Gram staining.

Moxifloxacin pharmacokinetics/pharmacodynamics and optimal dose and susceptibility breakpoint identification for treatment of disseminated Mycobacterium avium infection.

Organisms of the Mycobacterium avium-intracellulare complex (MAC) have been demonstrated to be susceptible to moxifloxacin. However, clinical data on how to utilize moxifloxacin to treat disseminated MAC are scanty. In addition, there have been no moxifloxacin pharmacokinetic-pharmacodynamic (PK/PD) studies performed for MAC infection. We utilized an in vitro PK/PD model of intracellular MAC to study moxifloxacin PK/PD for disseminated disease. Moxifloxacin doses, based on a serum half-life of 12 h, were administered, and the 0- to 24-h area under the concentration-time curve (AUC(0-24)) to MIC ratios associated with 1.0 log(10) CFU/ml per week kill and 90% of maximal kill (EC(90)) were identified. The AUC(0-24)/MIC ratio associated with 1.0 log(10) CFU/ml kill was 17.12, and that with EC(90) was 391.56 (r(2) = 0.97). Next, the moxifloxacin MIC distribution in 102 clinical isolates of MAC was identified. The median MIC was 1 to 2 mg/liter. Monte Carlo simulations of 10,000 patients with disseminated MAC were performed to determine the probability that daily moxifloxacin doses of 400 and 800 mg/day would achieve or exceed 1.0 log(10) CFU/ml per week kill or EC(90). Doses of 400 and 800 mg/day achieved the AUC(0-24)/MIC ratio of 17.12 in 64% and 92% of patients, respectively. The critical concentration of moxifloxacin against MAC was identified as 0.25 mg/liter in Middlebrook media. The proposed susceptibility breakpoint means that a larger proportion of clinical isolates is resistant to moxifloxacin prior to therapy. For patients infected with susceptible isolates, however, 800 mg a day should be examined for safety and efficacy for disseminated M. avium disease.

Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium monoazide.

One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false-positive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA-binding agent propidium monoazide (PMA) could distinguish viable from heat-inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic-treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C(t) difference consistently >3; p < 0.05) and after 10 days of treatment (C(t) difference >4; p < 0.01), when compared to viable cells (C(t) difference 1-2). Vancomycin had a stronger effect on the C(t) value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false-positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two-stage revision for infected arthroplasty.

Spectra MRSA, a new chromogenic agar medium to screen for methicillin-resistant Staphylococcus aureus.

A novel chromogenic medium, Spectra MRSA (Remel, Lenexa, KS), was designed to detect methicillin-resistant Staphylococcus aureus (MRSA) rapidly and more efficiently than traditional media (i.e., tryptic soy agar with 5% sheep blood [SBA] and mannitol salt agar [MSA]). A multicenter study (including four clinical trial sites and the Medical College of Wisconsin [MCW] Milwaukee, WI) compared the performance characteristics of Spectra MRSA to those of the traditional media for the detection of MRSA. For this study, 767 nasal swab specimens from the multicenter study (traditional medium used, SBA) and 667 nasal swab specimens from MCW (traditional medium used, MSA) were plated on each test medium and examined after 24 and 48 h of incubation. At 24 h, the sensitivity and the specificity of each medium were as follows: in the multicenter study, 95.4% and 99.7%, respectively, for Spectra MRSA and 93.6% and 100%, respectively, for SBA; at MCW, 95.2% and 99.5%, respectively, for Spectra MRSA and 88.7% and 94.0%, respectively, for MSA. The positive predictive values of each medium at 24 h were as follows: in the multicenter study, 98.1% for Spectra MRSA and 100% for SBA; at MCW, 95.2% for Spectra MRSA and 60.4% for MSA. In our evaluation, we found that Spectra MRSA was able to rapidly identify and differentiate methicillin-resistant S. aureus from methicillin-susceptible S. aureus on the basis of the utilization of chromogens that result in denim blue colonies, thus eliminating the need for biochemical analysis and antimicrobial susceptibility testing. Extending the incubation beyond 24 h did not significantly improve the recovery of MRSA and resulted in decreased specificity.

Limiting false-positive polymerase chain reaction results: detection of DNA and mRNA to differentiate viable from dead bacteria.

We compared 2 methods for determining Escherichia coli viability in vitro. A 16S rDNA polymerase chain reaction (PCR) assay detected bacteria irrespective of viability. A groEL mRNA reverse transcriptase PCR was positive for 72 h but later became negative. Detecting mRNA holds promise but is tedious, and groEL may not be the best target.

Improving clinical significance of PCR: use of propidium monoazide to distinguish viable from dead Staphylococcus aureus and Staphylococcus epidermidis.

Molecular techniques, such as the polymerase chain reaction (PCR) have high sensitivity when used to diagnose infection, but may detect DNA, RNA, and proteins from dead, as well as viable, bacteria. Propidium monoazide (PMA) is a DNA binding agent, that has the ability to penetrate only dead cells with compromised membranes and has been used in conjunction with real-time PCR to distinguish intact from dead bacterial cells. In this study, intact, heat-inactivated (dead), and intact/dead admixed Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were treated with PMA or left untreated before DNA extraction. We quantified levels of 16S rDNA and tuf gene by real-time quantitative PCR (qPCR), to test the ability of PMA to distinguish intact from dead bacteria. Our results indicated that PMA inhibited detection of dead bacteria, and the qPCR results reflected the number of intact bacteria without being impacted by the presence of the dead bacteria. This approach of combining qPCR with and without PMA treatment has promise to limit false-positive PCR results when used to diagnose infections, but needs to be further validated in clinical samples.

Bilateral periprosthetic joint infection caused by Salmonella enterica serotype Enteritidis, and identification of Salmonella sp using molecular techniques.

Salmonella septic arthritis is rare. Our objective was to identify bacterial species from joint fluid using a broad-range real-time PCR and pyrosequencing technique. We describe a case of bilateral Salmonella enterica serotype Enteritidis infection of right and left total knee arthroplasties. DNA was extracted from the joint fluid of the left knee, amplified by PCR, and the amplicons were evaluated by pyrosequencing. The patient was treated with ciprofloxacin, and the polyethylene liners were replaced in both knees. The results of pyrosequencing detected a Salmonella species. To the best of our knowledge, this is the first report describing the detection of Salmonella in joint fluid by universal PCR followed by pyrosequencing.

Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA.

BACKGROUND: The emergence of bla(KPC)-containing Klebsiella pneumoniae (KPC-Kp) isolates is attracting significant attention. Outbreaks in the Eastern USA have created serious treatment and infection control problems. A comparative multi-institutional analysis of these strains has not yet been performed. METHODS: We analysed 42 KPC-Kp recovered during 2006-07 from five institutions located in the Eastern USA. Antimicrobial susceptibility tests, analytical isoelectric focusing (aIEF), PCR and sequencing of bla genes, PFGE and rep-PCR were performed. Results By in vitro testing, KPC-Kp isolates were highly resistant to all non-carbapenem beta-lactams (MIC(90)s >or= 128 mg/L). Among carbapenems, MIC(50/90)s were 4/64 mg/L for imipenem and meropenem, 4/32 mg/L for doripenem and 8/128 for ertapenem. Combinations of clavulanate or tazobactam with a carbapenem or cefepime did not significantly lower the MIC values. Genetic analysis revealed that the isolates possessed the following bla genes: bla(KPC-2) (59.5%), bla(KPC-3) (40.5%), bla(TEM-1) (90.5%), bla(SHV-11) (95.2%) and bla(SHV-12) (50.0%). aIEF of crude beta-lactamase extracts from these strains supported our findings, showing beta-lactamases at pIs of 5.4, 7.6 and 8.2. The mean number of beta-lactamases was 3.5 (range 3-5). PFGE demonstrated that 32 (76.2%) isolates were clonally related (type A). Type A KPC-Kp isolates (20 bla(KPC-2) and 12 bla(KPC-3)) were detected in each of the five institutions. rep-PCR showed patterns consistent with PFGE. CONCLUSIONS: We demonstrated the complex beta-lactamase background of KPC-Kp isolates that are emerging in multiple centres in the Eastern USA. The prevalence of a single dominant clone suggests that interstate transmission has occurred.

Ventriculoperitoneal shunt infection caused by Actinomyces neuii subsp. neuii.

Actinomyces neuii subsp. neuii is a rare isolate in clinical specimens. The organism was previously designated CDC coryneform group 1 and was renamed in 1994. A case of a ventriculoperitoneal shunt infection caused by this organism is described.

Recognition and treatment of neonatal community-associated MRSA pneumonia and bacteremia.

Community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) have recently emerged as a major cause of serious infections among older children and are now being seen in NICU patients. We present the case of a preterm infant with CA-MRSA necrotizing pneumonia and secondary bacteremia.

The hsp65 gene patterns of less common Mycobacterium and Nocardia spp. by polymerase chain reaction-restriction fragment length polymorphism analysis with capillary electrophoresis.

To rapidly identify Mycobacterium and Nocardia spp. without costly probes, we had implemented capillary electrophoresis (CE) in polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis to analyze their 65-kDa heat shock protein (hsp65) gene. The PCR-RFLP analysis with CE (PRACE) involved only one restriction enzyme, HaeIII, and a single electrophoretic separation less than 10 min. Full-range (10-200 bp) RFLP patterns of 12 less common Mycobacterium and 7 Nocardia spp. were investigated. A good agreement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from sequence analysis. Including hsp65 gene patterns of 12 Mycobacterium spp. published earlier, differentiation was distinct among 24 Mycobacterium and 7 Nocardia spp. Some closely related species exhibiting similar biochemical characteristics could be well discriminated by an extra HaeIII digestion site. Thus, PRACE offers a nonprobe alternative for rapid identification of various cultured Mycobacterium and Nocardia to the species level.

Rapid species determination of Nocardia keratitis using pyrosequencing technology.

PURPOSE: To describe a new technique, pyrosequencing, which allows for the rapid identification of Mycobacterium and Nocardia species. DESIGN: Interventional case report. METHODS: The medical records of a patient presenting with an infectious keratitis were reviewed. RESULTS: A case of Nocardia abscessus/arthrititis/asiatica keratitis was diagnosed in a young individual with the aid of pyrosequencing technology. Based on presumed antibiotic sensitivities, therapy with topical trimethoprim-sulfamethoxazole eyedrops was initiated, and the infection was cleared rapidly with minimal residual scarring. CONCLUSIONS: Pyrosequencing may be a useful tool in aiding the rapid diagnosis and treatment of ocular infections caused by slow-growing pathogens.

Bacterial culture isolates from hospitalized pediatric patients with conjunctivitis.

PURPOSE: To determine the spectrum of organisms causing acute bacterial conjunctivitis in hospitalized children at a tertiary care referral center. DESIGN: Retrospective chart review. METHODS: Charts of hospitalized children with positive conjunctival cultures were reviewed, and patients with clinical description of conjunctivitis were studied. RESULTS: One hundred and seven isolates from 59 patients were included in the study. The most common organisms cultured were coagulase-negative Staphylococcus (59.3% of patients), viridans Streptococcus (47.5%), and Staphylococcus aureus (20.3%). The type of organisms differed based on age, with S. aureus and Haemophilus influenzae being more common in nonneonates. Gram-negative bacilli and methicillin-resistant Staphylococcus species were more common in patients hospitalized longer than two days. CONCLUSIONS: The distribution of bacterial organisms causing acute bacterial conjunctivitis in our hospitalized children differs from that of previous reports in the outpatient setting. Conjunctival swabbing for culture and sensitivities before instituting empiric broad-spectrum antibiotic therapy may be useful.

The use of real-time polymerase chain reaction for rapid diagnosis of skeletal tuberculosis.

We identified Mycobacterium tuberculosis DNA using real-time polymerase chain reaction on a specimen from an osteolytic lesion of a femoral condyle, in which the frozen section demonstrated granulomas. The process was much more rapid than is possible with culture. The rapid detection of M tuberculosis and the concomitant exclusion of granulomatous disease caused by nontuberculous mycobacteria or systemic fungi are necessary to appropriately treat skeletal tuberculosis. The detection and identification of M tuberculosis by culture may require several weeks using traditional methods. The real-time polymerase chain reaction method used has been shown to be rapid and reliable, and is able to detect and differentiate both tuberculous and nontuberculous mycobacteria. Real-time polymerase chain reaction may become a diagnostic standard for the evaluation of clinical specimens for the presence of mycobacteria; this case demonstrates the potential utility of this assay for the rapid diagnosis of skeletal tuberculosis.

Evaluation of the LightCycler Staphylococcus M GRADE kits on positive blood cultures that contained gram-positive cocci in clusters.

We evaluated the Roche LightCycler Staphylococcus M(GRADE) kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.