blaIMP-27 on transferable plasmids in Proteus mirabilis and Providencia rettgeri.

OBJECTIVES: A carbapenem-resistant Providencia rettgeri (PR1) isolate was recovered from a wound infection in Missouri, USA. This isolate possessed an EDTA-inhibitable carbapenemase that was unidentified using the Xpert CARBA-R assay. Our objective was to elucidate the molecular determinant of carbapenem resistance in this isolate. We then sought to test the transmissibility of blaIMP-27 loci in clinical P. rettgeri and Proteus mirabilis isolates. METHODS: In October 2016 the novel ambler Class B carbapenemase blaIMP-27, was reported in two different Proteus mirabilis (PM185 and PM187) isolates. Broth mating assays for transfer of carbapenemase activity were performed for the three clinical isolates with recipient sodium azide-resistant Escherichia coli J53. Antibiotic susceptibility testing and phenotypic carbapenemase activity testing were performed on the clinical isolates, J53 and transconjugants using the Kirby-Bauer disc diffusion method according to CLSI guidelines. Plasmid DNA from PM187, PR1 and their transconjugants were used as input for Nextera Illumina sequencing libraries and sequenced on a NextSeq platform. RESULTS: PR1 was resistant to both imipenem and meropenem. PM187 and PR1 could transfer resistance to E. coli through plasmid conjugation (pPM187 and pPR1). pPM187 had a virB/virD4 type IV secretion system whereas pPR1 had a traB/traD type IV secretion system. CONCLUSION: Two of three blaIMP-27-bearing clinical isolates tested could conjugate resistance into E. coli. The resulting transconjugants became positive for phenotypic carbapenemase production but did not pass clinical resistance breakpoints. blaIMP-27 can be transmitted on different plasmid replicon types that rely on distinct classes of type IV secretion system for horizontal transfer.

Diagnostic accuracy of fungal identification in histopathology and cytopathology specimens.

Tools to diagnose fungal infection are microscopic examination, antigen or antibody-based detection tests, molecular diagnostics, and culture, with culture being the "gold standard" for species-level identification. Although these methods are commonly used in concert and yield concordant results, in some cases tissue is not available for culture, and/or different methodologies yield discrepant results. These discrepancies may be clinically significant, causing confusion and inappropriate or delayed initiation of antifungals. This study evaluates the correlation between microscopic examination and the results of laboratory studies, and identifies clinical scenarios and specimen characteristics associated with tissue sent for microscopic examination without concomitant laboratory studies. We performed an 18-year retrospective review at a tertiary-care, academic medical center in the Midwest United States of all fungal infection diagnoses made by microscopic examination. Only 16% of samples with fungal infection diagnosed by microscopic examination had a concomitant sample submitted for laboratory studies. Of these cases, 36% had no growth on culture and/or had a negative laboratory study. Among cases in which fungal infections were diagnosed and laboratory studies were positive, the accuracy of histopathologic identification was 95%. The most common cause for incorrect morphologic diagnoses was misidentification of Aspergillus spp. and Mucorales. Our results underscore the importance of educating pathologists with regard to appropriate terminology and increasing knowledge of mycology, particularly in relation to organisms forming hyphae in tissue. Species-level diagnosis of fungi cannot be made by microscopic examination of tissue alone. Anatomic pathology reports should recommend correlation with laboratory studies, and provide a differential diagnosis based on morphology.

Utility of strain typing of Propionibacterium acnes in central nervous system and prosthetic joint infections to differentiate contamination from infection: a retrospective cohort.

The study aimed to retrospectively assess if strain typing of Propionibacterium acnes could help to distinguish between infection and contamination in isolates recovered from the central nervous system (CNS) and prosthetic joints (PJs). This was a retrospective cohort of all Propionibacterium species isolates from the Barnes-Jewish Hospital (St Louis, MO, USA) clinical microbiology laboratory from 2011 to 2014. Available frozen isolates were recovered, and strain type (IA-1, IA-2, IB, II, III, or nontypeable class A or B) was determined via polymerase chain reaction (PCR)-based methods. For CNS isolates, P. acnes was considered pathogenic if treating physicians administered ≥7 days of directed antibiotic therapy against P. acnes. During the study period, Propionibacterium species was isolated from clinical cultures 411 times. 152 isolates were available for analysis. Of the 152 isolates, 140 were confirmed to be P. acnes, 61 of which were from the CNS (45 contaminants, 16 infections). Strain type IA-1 was more common (50.0%, 8 out of 16) among CNS infections than among contaminants (22.2%, 10 out of 45). For PJ isolates 61.3% (19 out of 31) met the criteria for infection. The predominant strain type for CNS infection was IA-1 and for PJ isolates, IB. Strain type IA-1 was isolated more often in patients with CNS infections, which may indicate a predilection of this strain type to cause CNS infection. Future research should prospectively evaluate strain typing as a means of assisting in the diagnosis of CNS infections and confirm our findings.

Rapid MRSA PCR on respiratory specimens from ventilated patients with suspected pneumonia: a tool to facilitate antimicrobial stewardship.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of pneumonia in ventilated patients. Our objective was to evaluate the GeneXpert MRSA/SA SSTI Assay (Xpert MRSA/SA) (Cepheid, Sunnyvale, CA) for use in lower respiratory tract (LRT) specimens for rapid MRSA detection and to determine the potentially saved antibiotic-days if a culture-based identification method was replaced by this assay. Remnant LRT samples from ventilated patients submitted to the microbiology laboratory for routine culture were tested using conventional culture and Xpert MRSA/SA. One hundred of 310 LRT specimens met the inclusion criteria. Ten samples were positive for MRSA by Xpert MRSA/SA, while six were positive by routine culture methods. Xpert MRSA/SA correctly identified 5/6 positive and 89/94 negative MRSA specimens, for a sensitivity of 83.3%, specificity of 94.7%, positive predictive value of 45.6%, and negative predictive value of 98.9%. The assay also correctly detected 3/3 positive and 90/97 negative methicillin-susceptible S. aureus (MSSA) specimens, for a sensitivity of 100%, specificity of 92.8%, positive predictive value of 30%, and negative predictive value of 100%. A total of 748 vancomycin and 305 linezolid antibiotic-days were associated with the enrolled specimens. Vancomycin and linezolid utilization could decrease by 68.4% and 83%, respectively, if discontinued 1 day after negative polymerase chain reaction (PCR) results. The Xpert MRSA/SA SSTI rapid MRSA PCR assay performed well in respiratory samples from ventilated patients with suspected pneumonia and has the potential to facilitate stewardship efforts such as reducing empiric vancomycin and linezolid therapy.

Emergence of community-associated methicillin-resistant Staphylococcus aureus strains in the neonatal intensive care unit: an infection prevention and patient safety challenge.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause significant morbidity and mortality in neonatal intensive care units (NICUs). We characterized the clinical and molecular epidemiology of MRSA strains colonizing NICU patients. Nasal MRSA isolates (n = 250, from 96 NICU patients) recovered through active surveillance from 2009 to 2014 were characterized with staphylococcal cassette chromosome mec (SCCmec) typing and detection of mupA (marker of high-level mupirocin resistance) and qacA/B (marker associated with chlorhexidine resistance). Factors associated with community-associated (CA-) or healthcare-associated (HA-) MRSA were evaluated. The overall prevalence of MRSA nasal colonization was 3.9%. Of 96 neonates in our retrospective cohort, 60 (63%) were colonized with CA-MRSA strains and 35 (36%) were colonized with HA-MRSA strains. Patients colonized with HA-MRSA were more likely to develop MRSA infections than patients colonized with CA-MRSA (13/35, 37% versus 8/60, 13%; p 0.007), although the interval from colonization to infection was shorter in CA-MRSA-colonized infants (median 0 days, range -1 to 4 versus HA-MRSA-colonized infants, 7 days, -1 to 43; p 0.005). Maternal peripartum antibiotics were associated with CA-MRSA colonization (adjusted odds ratio (aOR) 8.7; 95% CI 1.7-45.0); intubation and surgical procedures were associated with HA-MRSA colonization (aOR 7.8; 95% CI 1.3-47.6 and aOR 6.0; 95% CI 1.4-24.4, respectively). Mupirocin- and chlorhexidine-resistant MRSA was isolated from four and eight patients, respectively; carriage of a mupirocin-resistant strain precluded decolonization. CA-MRSA strains are prominent in the NICU and associated with distinct risk factors. Given community reservoirs for MRSA acquisition and transmission, novel infection prevention strategies are needed.

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group.

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.

Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively.

Rapid emergence of daptomycin resistance in clinical isolates of Corynebacterium striatum a cautionary tale.

The objective of this study was to investigate the observation of daptomycin resistance in Corynebacterium striatum, both in vivo and in vitro. We describe a case of C. striatum bacteremia in a patient with a left ventricular assist device (LVAD); the initial isolate recovered was daptomycin susceptible with a minimum inhibitory concentration (MIC) of 0.125 µg/ml. Two months later, and after daptomycin therapy, the individual became bacteremic with an isolate of C. striatum with a daptomycin MIC of >256 µg/ml. To study the prevalence of daptomycin resistance in C. striatum, clinical isolates of C. striatum were grown in broth culture containing daptomycin to investigate the emergence of resistance to this antimicrobial. Molecular typing was used to evaluate serial isolates from the index patient and the clinical isolates of C. striatum we assayed. In vitro analysis of isolates from the index patient and 7 of 11 additional C. striatum isolates exhibited the emergence of high-level daptomycin resistance, despite initially demonstrating low MICs to this antimicrobial agent. This phenotype was persistent even after serial subculture in the absence of daptomycin. Together, these data demonstrate that caution should be taken when using daptomycin to treat high-inoculum infections and/or infections of indwelling medical devices with C. striatum. To our knowledge, this is the first report characterizing the emergence of daptomycin resistance in C. striatum.

Evaluation of the Bruker Biotyper and VITEK MS MALDI-TOF MS systems for the identification of unusual and/or difficult-to-identify microorganisms isolated from clinical specimens.

The purpose of this investigation was to evaluate the analytical performance characteristics of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of unusual organisms. We evaluated the accuracy of two MALDI-TOF MS systems, bioMérieux VITEK MS (database v2.0) and Bruker Biotyper (software version 3.0), for the identification of the most difficult and/or unusual microorganisms isolated from clinical specimens. Our study included 174 bacterial isolates recovered from clinical cultures at Barnes-Jewish Hospital, St. Louis, MO, from 2009 to 2013, representing 50 genera and 52 species. MS identifications were compared to the identification reported by the reference laboratory. Discrepancies were resolved using molecular methods, including 16S rRNA gene sequencing and additional molecular methods. When performed, molecular methods were considered the gold standard. Of the 168 isolates resolved to the genus level, VITEK MS identified 145 (86.3 %), and of the 114 isolates resolved to the species level, 97 (85.1 %) were correctly identified. Bruker Biotyper identified 155 (92.3 %) of 168 isolates to the genus level and 97 (85.1 %) of 114 isolates to the species level. VITEK MS and Bruker Biotyper provided no identification for 17 (10.1 %) and 12 (7.1 %) organisms, respectively, and misidentified six (3.6 %) and one (0.6 %) isolate, respectively. Six isolates (3.6 %) were not resolvable to the genus level and were excluded from data analysis due to the lack of a gold standard for comparison. There was no significant difference in the number of organisms identified to the genus level, species level, unidentified, or misidentified by the two MALDI-TOF MS systems (p = 0.11, 1.0, 0.44, and 0.12, respectively).

Comparison and optimization of two MALDI-TOF MS platforms for the identification of medically relevant yeast species.

The rapid identification of yeast is essential for the optimization of antifungal therapy. The objective of our study was to evaluate two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms, the bioMérieux VITEK MS (IVD Knowledgebase v.2.0) and Bruker Biotyper (software version 3.1), for the rapid identification of medically relevant yeast. One hundred and seventeen isolates, representing six genera and 18 species, were analyzed using multiple direct smear methods to optimize identification. Sequence analysis was the gold standard for comparison. Isolates were analyzed with VITEK MS using the direct smear method +/- a 25 % formic acid on-plate extraction. For Biotyper, isolates were analyzed using direct smear without formic acid, and with 25 % and 100 % formic acid on-plate extractions. When all methods were included, VITEK MS correctly identified 113 (96.6 %) isolates after 24 h with one misidentification, and Biotyper correctly identified 77 (65.8 %) isolates using a threshold of ≥2.0 with no misidentifications. Using a revised threshold of ≥1.7, Biotyper correctly identified 103 (88.0 %) isolates, with 3 (2.6 %) misidentifications. For both platforms, the number of identifications was significantly increased using a formic acid overlay (VITEK MS, p < 0.01; Biotyper, p < 0.001), and reducing the Biotyper threshold from ≥2.0 to ≥1.7 significantly increased the rate of identification (p < 0.001). The data in this study demonstrate that the direct smear method with on-plate formic acid extraction can be used for yeast identification on both MS platforms, and more isolates are identified using the VITEK MS system (p < 0.01).

Multi-center evaluation of the VITEK® MS system for mass spectrometric identification of non-Enterobacteriaceae Gram-negative bacilli.

Studies have demonstrated that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate method for the identification of clinically relevant bacteria. The purpose of this study was to evaluate the performance of the VITEK MS v2.0 system (bioMérieux) for the identification of the non-Enterobacteriaceae Gram-negative bacilli (NEGNB). This multi-center study tested 558 unique NEGNB clinical isolates, representing 18 genera and 33 species. Results obtained with the VITEK MS v2.0 were compared with reference 16S rRNA gene sequencing and when indicated recA sequencing and phenotypic analysis. VITEK MS v2.0 provided an identification for 92.5 % of the NEGNB isolates (516 out of 558). VITEK MS v2.0 correctly identified 90.9 % of NEGNB (507 out of 558), 77.8 % to species level and 13.1 % to genus level with multiple species. There were four isolates (0.7 %) incorrectly identified to genus level and five isolates (0.9 %), with one incorrect identification to species level. The remaining 42 isolates (7.5 %) were either reported as no identification (5.0 %) or called "mixed genera" (2.5 %) since two or more different genera were identified as possible identifications for the test organism. These findings demonstrate that the VITEK MS v2.0 system provides accurate results for the identification of a challenging and diverse group of Gram-negative bacteria.

Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l'Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer's instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7% of the 965 isolates tested, with 83.8% correct to the species level and 12.8% limited to a genus-level identification. There was no identification for 1.7% of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

The ecology and pathobiology of Clostridium difficile infections: an interdisciplinary challenge.

Clostridium difficile is a well recognized pathogen of humans and animals. Although C. difficile was first identified over 70 years ago, much remains unknown in regards to the primary source of human acquisition and its pathobiology. These deficits in our knowledge have been intensified by dramatic increases in both the frequency and severity of disease in humans over the last decade. The changes in C. difficile epidemiology might be due to the emergence of a hypervirulent stain of C. difficile, ageing of the population, altered risk of developing infection with newer medications, and/or increased exposure to C. difficile outside of hospitals. In recent years, there have been numerous reports documenting C. difficile contamination of various foods, and reports of similarities between strains that infect animals and strains that infect humans as well. The purposes of this review are to highlight the many challenges to diagnosing, treating, and preventing C. difficile infection in humans, and to stress that collaboration between human and veterinary researchers is needed to control this pathogen.