Ainsley C. Nicholson

Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States

ABSTRACT USA300 methicillin-resistant Staphylococcus aureus (MRSA) isolates are usually resistant only to oxacillin, erythromycin, and, increasingly, levofloxacin. Of these, oxacillin and levofloxacin resistances are chromosomally encoded. Plasmid-mediated clindamycin, mupirocin, and/or tetracycline resistance has been observed among USA300 isolates, but these descriptions were limited to specific patient populations or isolated occurrences. We examined the antimicrobial susceptibilities of invasive MRSA isolates from a national surveillance population in order to identify USA300 isolates with unusual, possibly emerging, plasmid-mediated antimicrobial resistance. DNA from these isolates was assayed for the presence of resistance determinants and the presence of a pSK41-like conjugative plasmid. Of 823 USA300 isolates, 72 (9%) were tetracycline resistant; 69 of these were doxycycline susceptible and tetK positive, and 3 were doxycycline resistant and tetM positive. Fifty-one (6.2%) isolates were clindamycin resistant and ermC positive; 22 (2.7%) isolates were high-level mupirocin resistant (mupA positive); 5 (0.6%) isolates were trimethoprim-sulfamethoxazole (TMP-SMZ) resistant, of which 4 were dfrA positive; and 7 (0.9%) isolates were gentamicin resistant and aac6′-aph2″ positive. Isolates with pSK41-like plasmids (n = 24) were positive for mupA (n = 19), dfrA (n = 6), aac6′-aph2″ (n = 6), tetM (n = 2), and ermC (n = 8); 20 pSK41-positive isolates were positive for two or more resistance genes. Conjugative transfer of resistance was demonstrated between four gentamicin- and mupirocin-resistant and three gentamicin- and TMP-SMZ-resistant USA300 isolates; transconjugants harbored a single pSK41-like plasmid, which was PCR positive for aac6′-aph2″ and either mupA and/or dfrA. USA300 and USA100 isolates from the same state with identical resistance profiles contained pSK41-like plasmids with indistinguishable restriction and Southern blot profiles, suggesting horizontal plasmid transfer between USA100 and USA300 isolates.

Complete Restriction of Fluoroquinolone Use to Control an Outbreak of Clostridium difficile Infection at a Community Hospital

OBJECTIVE To review the effect of interventions, including a complete restriction in the use of fluoroquinolones (FQs), used to control an outbreak of hospital-onset Clostridium difficile infection (HO-CDI) caused primarily by the epidemic North American pulsed-field gel electrophoresis type 1 strain. DESIGN Retrospective cohort and case-control study of all episodes of HO-CDI both before and after 2 interventions. SETTING Community hospital; January 1, 2005, through March 31, 2007. Interventions. Complete, 5-month, facility-wide restriction of fluoroquinolone use, during which a change in the environmental-services contractor occurred. RESULTS During a 27-month period, 319 episodes of HO-CDI occurred. The hospital-wide mean defined daily doses of antimicrobials decreased 22% after restricting FQ use, primarily because of a 66% decrease in the use of FQs. The interventions were also associated with a significant change in the HO-CDI incidence trends and with an absolute decrease of 22% in HO-CDI cases caused by the epidemic strain (from 66% before the intervention period to 44% during and after the intervention period; P=.02). Univariate analysis revealed that case patients with HO-CDI due to the epidemic strain were more likely than control patients, who did not have diarrhea, to receive a FQ, whereas case patients with HO-CDI due to a nonepidemic strain were not. However, FQ use was not significantly associated with HO-CDI in multivariable analysis. CONCLUSIONS An outbreak of epidemic-strain HO-CDI was controlled at a community hospital after an overall decrease in antimicrobial use, primarily because of a restriction of FQ use and a change in environmental-services contractors. The restriction of FQ use may be useful as an adjunct control measure in a healthcare facilities during outbreaks of epidemic-strain HO-CDI.

Major families of multiresistant plasmids from geographically and epidemiologically diverse staphylococci.

Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20–30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.

Scardovia wiggsiae sp. nov., isolated from the human oral cavity and clinical material, and emended descriptions of the genus Scardovia and Scardovia inopinata

Six strains of anaerobic, pleomorphic Gram-positive bacilli, isolated from the human oral cavity and an infected arm wound, were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. 16S rRNA gene sequence analysis revealed that the isolates were most closely related to Scardovia inopinata CCUG 35729(T) (94.8-94.9 % 16S rRNA gene sequence similarity). The isolates were saccharolytic and produced acetic and lactic acids as end products of fermentation. The major fatty acids were C(16 : 0) (49.8 %) and C(18 : 1)ω9c (35.8 %). Polar lipid analysis revealed a variety of glycolipids, diphosphatidylglycerol, an unidentified phospholipid and an unidentified phosphoglycolipid. No respiratory quinones were detected. The peptidoglycan was of the type A4α L-Lys-Thr-Glu, with L-lysine partially replaced by L-ornithine. The DNA G+C content of one of the strains, C1A_55(T)(,) was 55 mol%. A novel species, Scardovia wiggsiae sp. nov., is proposed to accommodate the six isolates, with the type strain C1A_55(T) (=DSM 22547(T)=CCUG 58090(T)).

DNA–DNA hybridization study of strains of Chryseobacterium, Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc, IIe, IIh and IIi, mostly from human clinical sources, and proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov

The taxonomic classification of 182 phenotypically similar isolates was evaluated using DNA-DNA hybridization and 16S rRNA gene sequence analysis. These bacterial isolates were mainly derived from clinical sources; all were Gram-negative non-fermenters and most were indole-producing. Phenotypically, they resembled species from the genera Chryseobacterium, Elizabethkingia or Empedobacter or belonged to CDC groups IIc, IIe, IIh and IIi. Based on these analyses, four novel species are described: Chryseobacterium bernardetii sp. nov. (type strain NCTC 13530(T) = CCUG 60564(T) = CDC G229(T)), Chryseobacterium carnis sp. nov. (type strain NCTC 13525(T) = CCUG 60559(T) = CDC G81(T)), Chryseobacterium lactis sp. nov. (type strain NCTC 11390(T) = CCUG 60566(T) = CDC KC1864(T)) and Chryseobacterium nakagawai sp. nov. (type strain NCTC 13529(T) = CCUG 60563(T) = CDC G41(T)). The new combination Chryseobacterium taklimakanense comb. nov. (type strain NCTC 13490(T) = X-65(T) = CCTCC AB 208154(T) = NRRL B-51322(T)) is also proposed to accommodate the reclassified Planobacterium taklimakanense.

Multiplex Real-Time PCR Assay for Detection of Methicillin-Resistant Staphylococcus aureus and Associated Toxin Genes

ABSTRACT We describe a real-time PCR assay for the detection of methicillin-resistant Staphylococcus aureus and genes encoding toxic shock syndrome toxin 1 and Panton-Valentine leukocidin. Rapid screening and detection of toxins is a useful tool for surveillance studies and outbreak investigations involving large numbers of isolates.

Nocardia amikacinitolerans sp. nov., an amikacin-resistant human pathogen

Five nocardioform isolates from human clinical sources were evaluated. Analysis of the nearly full-length 16S rRNA gene showed 99.9-100 % similarity among the strains. The results of a comparative phylogenetic analysis of the 16S rRNA gene sequences indicated that the isolates belonged to the genus Nocardia. Phenotypic and molecular analyses were performed on the clinical isolates. Traditional phenotypic analyses included morphological, biochemical/physiological, chemotaxonomic and antimicrobial susceptibility profiling. Molecular studies included 1441-bp 16S rRNA and 1246-bp gyrB gene sequence analyses, as well as DNA-DNA hybridizations. Biochemical analysis failed to differentiate the putative novel species from its phylogenetic neighbours; however, molecular studies were able to distinguish the patient strains and confirm them as members of a single species. Based on 16S rRNA gene sequence analysis, similarity between the isolates and their closest relatives (type strains of Nocardia araoensis, N. arthritidis, N. beijingensis and N. niwae) was ≤99.3 %. Analysis of partial gyrB gene sequences showed 98-99.7 % relatedness among the isolates. Nocardia lijiangensis and N. xishanensis were the closest related species to the isolates based on gyrB gene sequence analysis, and their type strains showed 95.7 and 95.3 % similarity, respectively, to strain W9988(T). Resistance to amikacin and molecular analyses, including DNA-DNA hybridization, distinguished the five patient strains from their phylogenetic neighbours, and the results of this polyphasic study indicated the existence of a novel species of Nocardia, for which we propose the name Nocardia amikacinitolerans sp. nov., with strain W9988(T) ( = DSM 45539(T)  = CCUG 59655(T)) as the type strain.

Evolutionary dynamics and genomic features of the Elizabethkingia anophelis 2015 to 2016 Wisconsin outbreak strain

An atypically large outbreak of Elizabethkingia anophelis infections occurred in Wisconsin. Here we show that it was caused by a single strain with thirteen characteristic genomic regions. Strikingly, the outbreak isolates show an accelerated evolutionary rate and an atypical mutational spectrum. Six phylogenetic sub-clusters with distinctive temporal and geographic dynamics are revealed, and their last common ancestor existed approximately one year before the first recognized human infection. Unlike other E. anophelis, the outbreak strain had a disrupted DNA repair mutY gene caused by insertion of an integrative and conjugative element. This genomic change probably contributed to the high evolutionary rate of the outbreak strain and may have increased its adaptability, as many mutations in protein-coding genes occurred during the outbreak. This unique discovery of an outbreak caused by a naturally occurring mutator bacterial pathogen provides a dramatic example of the potential impact of pathogen evolutionary dynamics on infectious disease epidemiology.

Draft Genome Sequences of Strains Representing Each of the Elizabethkingia Genomospecies Previously Determined by DNA-DNA Hybridization

ABSTRACT Draft genome sequences of Elizabethkingia meningoseptica and representatives of each of its four historically described genomospecies were sequenced here. Preliminary analysis suggests that Elizabethkingia miricola belongs to genomospecies 2, and both Elizabethkingia anophelis and Elizabethkingia endophytica are most similar to genomospecies 1.

Correlation of psycho-neuroendocrine-immune (PNI) gene expression with symptoms of acute infectious mononucleosis.

Acute infection is known to perturb psycho-neuroendocrine-immune (PNI) gene expression. Oligonucleotide microarrays were used to examine PNI gene expression in the peripheral blood of 13 subjects with infectious mononucleosis (IM). Novel peripheral blood gene expression activity was correlated with central-nervous-system-mediated symptoms including fatigue and sleep disturbance. Of note, expression of the MADS box transcription enhancer factor 2 polypeptide C (MEF2C) gene, previously implicated in skeletal muscle myogenesis, correlated with symptoms of musculo-skeletal pain and fatigue. Expression of the hypocretin/orexin receptor HCRTR2, which has been implicated in narcolepsy, correlated with sleep disturbance. And, VACHT, the vesicular acetylcholine transporter, was highly correlated with neurocognitive disturbance. The expression of both HCRTR2 and MEF2C in the peripheral blood was validated by reverse transcription PCR. Thus, investigation of the PNI response in peripheral blood may provide novel insights into the complex pathophysiology of centrally mediated disease states.