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.

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.

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.

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.

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.

Comparison of traditional phenotypic identification methods with partial 5' 16S rRNA gene sequencing for species-level identification of nonfermenting Gram-negative bacilli.

Correct identification of nonfermenting Gram-negative bacilli (NFB) is crucial for patient management. We compared phenotypic identifications of 96 clinical NFB isolates with identifications obtained by 5' 16S rRNA gene sequencing. Sequencing identified 88 isolates (91.7%) with >99% similarity to a sequence from the assigned species; 61.5% of sequencing results were concordant with phenotypic results, indicating the usability of sequencing to identify NFB.

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.

Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles.

We evaluated the performance of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata, in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C. albicans) or fluorescent red (C. glabrata) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available <3 h after cultures signal positive. An evaluation of 197 routine blood culture bottles newly positive for yeast by Gram staining was performed at five hospitals. The sensitivities of detection for C. albicans, and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.

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.

Pyrosequencing as a tool for the identification of common isolates of Mycobacterium sp.

Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.

Rotaviral and bacterial gastroenteritis in children during winter: an evaluation of physician ordering patterns.

BACKGROUND: Identification of the agents of infectious diarrhea may facilitate appropriate therapy and prevent inappropriate antibiotic use. OBJECTIVES: To better define the etiology of infectious diarrhea for children <12 years in our community and to study the ordering patterns of physicians. STUDY DESIGN: We reviewed test results of stool specimens from children <12 years old at our institution (CCF) and those submitted through our reference laboratory for rotavirus enzyme immunoassay (REIA) and stool cultures for a 7-month period (11/1/00-6/1/01). For CCF patients, REIA and stool cultures for usual bacterial enteric pathogens (BEP) were performed, regardless of the test ordered (i.e. REIA alone, stool culture alone or both). We compared the results with the orders placed to determine if requests for rotavirus alone or bacterial stool culture alone missed BEP or rotavirus, respectively. RESULTS: Overall, REIAs were performed on 81% (538/661) of stool specimens, with 37% positive. Stool cultures were performed on 62% (408/661) of stool specimens, with 4.4% positive. Stool specimens (280) from CCF pediatric patients were evaluated for both rotavirus and BEP. Some 42% of REIA and 23% of stool cultures were ordered as single tests, while both tests were ordered for 35% of the patients. Of the REIA ordered alone, 34% were positive for rotavirus; however, 2.5% of these contained BEP that would have been missed. Of the stool cultures that were ordered alone, 8% were positive; however, 19% of these contained rotavirus that would have been missed. When both tests were ordered, 22% contained rotavirus and 2% contained BEP. CONCLUSION: Both rotavirus and bacterial enteric infections were missed with selective viral versus bacterial specific ordering patterns. A rotaviral screen prior to stool culture may be useful for children with diarrhea during the winter months.

Cross-class resistance to non-beta-lactam antimicrobials in extended-spectrum beta-lactamase-producing Klebsiella pneumoniae.

Extended spectrum beta-lactamases are modified beta-lactamase enzymes that impart resistance to third-generation cephalosporins and make all beta-lactam antibiotics and cephalosporins useless for therapy. We compared the antimicrobial susceptibility profiles of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing isolates of Klebsiella pneumoniae. The ESBL producers had significantly diminished susceptibility compared with the non-ESBL producers for gentamicin (P < .001), tobramycin (P < .001), amikacin (P < .005), trimethoprim-sulfamethoxazole (P < .01), ciprofloxacin (P < .001), and nitrofurantoin (P < .001). All isolates were susceptible to imipenem. ESBL-producing K pneumoniae may also be resistant to non-beta-lactam antibiotics. Therefore, susceptibility testing of these isolates is critical for guiding therapy.

Microbiology of liver abscesses and the predictive value of abscess gram stain and associated blood cultures.

Although rare, pyogenic liver abscesses are potentially fatal. We evaluated the predictive value of Gram stain of liver abscess aspirates and temporally associated blood cultures. Gram stains detected bacteria in 79% of the liver abscesses tested. The sensitivity and specificity of Gram stain of the liver abscesses were 90% and 100% for Gram-positive cocci (GPC) and 52% and 94% for Gram-negative bacilli (GNB). The sensitivities of the blood cultures for any GPC and GNB present in the liver abscess were 30% and 39%, respectively. Although, Gram stains and blood cultures offer incomplete detection of the microbial contents of pyogenic liver abscesses, both tests should always accompany liver abscess cultures.

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.

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.

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.