Sanjay K. Shukla

A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus.

Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994), and that expansion of the European clade began in 1995 (1991 to 1999), several years before the new clone was recognized. Demographic analysis based on DNA sequence variation indicated a sharp increase of bacterial population size from 2001 to 2004, which is concordant with the reported prevalence of this strain in several European countries. A detailed ancestry-based reconstruction of the spatiotemporal dispersal dynamics suggested a pattern of frequent transmission of the ST225 clone among hospitals within Central Europe. In addition, comparative genomics indicated complex bacteriophage dynamics.

Addition of ceftaroline to daptomycin after emergence of daptomycin-nonsusceptible Staphylococcus aureus during therapy improves antibacterial activity.

ABSTRACT Antistaphylococcal beta-lactams enhance daptomycin activity and have been used successfully in combination for refractory methicillin-resistant Staphylococcus aureus (MRSA) infections. Ceftaroline possesses MRSA activity, but it is unknown if it improves the daptomycin potency comparably to other beta-lactams. We report a complex patient case of endocarditis who was treated with daptomycin in combination with ceftaroline, which resulted in clearance of a daptomycin-nonsusceptible strain. An in vitro pharmacokinetic/pharmacodynamic model of renal failure was used to simulate the development of daptomycin resistance and evaluate the microbiologic effects of daptomycin plus ceftaroline treatment. Combination therapy with daptomycin and ceftaroline restored daptomycin sensitivity in vivo and resulted in clearance of persistent blood cultures. Daptomycin susceptibility in vitro was increased in the presence of either ceftaroline or oxacillin. Daptomycin at 6 mg/kg of body weight every 48 h was bactericidal in the model but resulted in regrowth and daptomycin resistance (MIC, 2 to 4 μg/ml) with continued monotherapy. The addition of ceftaroline at 200 mg every 12 h after the emergence of daptomycin resistance enhanced bacterial killing. Importantly, daptomycin plus ceftaroline as the initial combination therapy produced rapid and sustained bactericidal activity and prevented daptomycin resistance. Both in vivo- and in vitro-derived daptomycin resistance resulted in bacteria with more fluid cell membranes. After ceftaroline was added in the model, fluidity was restored to the level of the initial in vivo isolate. Daptomycin-resistant isolates required high daptomycin exposures (at least 10 mg/kg) to optimize cell membrane damage with daptomycin alone. Ceftaroline combined with daptomycin was effective in eliminating daptomycin-resistant MRSA, and these results further justify the potential use of daptomycin plus beta-lactam therapy for these refractory infections.

Genetic variants of Anaplasma phagocytophilum infecting dogs in Western Washington State.

ABSTRACT Eight dogs from western Washington State suspected of being infected with Anaplasma phagocytophilum because of the finding of morulae in peripheral blood neutrophils were studied for determination of the etiologic agent of disease. All cases were diagnosed between April 2003 and April 2004. Six of the eight dogs had no travel history during the 6 months prior to presentation. Two dogs had traveled within the Northwest United States and Canada. Fever, lethargy, and anorexia were the most common clinical signs in the dogs. Lymphopenia, thrombocytopenia, and an elevated activity of alkaline phosphatase in the serum were the most common laboratory findings. All dogs tested during the acute phase of clinical signs were seropositive for A. phagocytophilum antibodies but negative for Ehrlichia canis antibodies. PCR amplification and direct sequencing of portions of the 16S rRNA gene from the whole blood of all seven dogs that were tested yielded A. phagocytophilum after a comparison to bacterial sequences available in the GenBank database. Five genetic variants were identified based on one or two nucleotide differences in the 16S rRNA gene sequences at nucleotide positions 54, 84, 86, and 120. Individual dogs were infected with more than one variant. Treatment with doxycycline or tetracycline resulted in a rapid resolution of clinical signs. The occurrence of canine granulocytic anaplasmosis in western Washington State suggests that A. phagocytophilum infection should be considered in differential diagnoses of dogs presenting with lethargy, anorexia, fever, and lameness, particularly in the context of lymphopenia, thrombocytopenia, and increased serum alkaline phosphatase. The zoonotic importance of A. phagocytophilum should support an increase in surveillance for horses and people residing in this area.

Molecular Characteristics of Nosocomial and Native American Community-Associated Methicillin-Resistant Staphylococcus aureus Clones from Rural Wisconsin

ABSTRACT In central and northern Wisconsin methicillin-resistant Staphylococcus aureus (MRSA) was first detected in 1989. Over the next 10-year period, 581 MRSA isolates were collected, 17.2% of which came from patients who were treated at five Native American clinics. These isolates were typed by SmaI-macrorestricted pulsed-field gel electrophoresis (PFGE). The PFGE patterns clustered the isolates into six major clonal groups (MCGs), i.e., MCGs 1 to 6, and 19 minor clonal groups (mCGs). The 25 clonal groups were represented by 109 unique PFGE types. Sixty-five percent of the MCG-2 isolates were recovered from patients who were treated at Native American clinics. Ninety-four percent of the MCG-2 isolates harbored the staphylococcal cassette chromosome mec (SCCmec) IVa. These isolates also had PFGE profiles that were clonally related to the midwestern community-associated MRSA (CA-MRSA) strain, MW2. The representative isolates from MCG-2 had the multilocus sequence type allelic profile 1-1-1-1-1-1-1 and contained pvl genes. They were also susceptible to various antibiotics, a finding consistent with the CA-MRSA phenotype. SCCmec IV was also present in other mCGs. Unlike MCG-2, isolates from the remaining five MCGs harbored SCCmec II and were resistant to multiple antibiotics, suggesting their nosocomial origin. The 19 mCGs were represented by diverse SCCmec types and three putative new variants referred to as SCCmec Ib, IIa, and IIb.

Evidence of Multiple Virulence Subtypes in Nosocomial and Community-Associated MRSA Genotypes in Companion Animals from the Upper Midwestern and Northeastern United States

Objective: Not much is known about the zoonotic transmission of methicillin-resistant Staphylococcus aureus (MRSA) in companion animals in the United States. We report the rate of prevalence of S. aureus and MRSA recovered from clinical samples of animals requiring treatment at veterinary clinics throughout the upper midwestern and northeastern United States. Design: We compared phenotypes, genotypes, and virulence profiles of the MRSA isolates identified in companion animals, such as cats, dogs, horses, and pigs, with typical human nosocomial and community-associated MRSA (CA-MRSA) genotypes to assess implied zoonotic transmission or zooanthroponosis. Five hundred thirty-three coagulase-positive staphylococci (CPS) isolates recovered between 2006 and 2008 from a variety of animal-source samples were screened for S. aureus by S. aureus-specific 16S rDNA primers and were screened for methicillin-resistance. All MRSA isolates were genotyped by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa typing. They were also screened for common staphylococcal enterotoxin and adhesion genes by multiplex and singleplex PCR. Results: Among the 533 CPS isolates recovered, 66 (12.4%) were determined to be S. aureus and 24 (4.5%) were MRSA. The percent of animals that were positive for S. aureus were as follows: 6.6% (32 of 487) dogs, 39.6% (19 of 48) cats, 83.3% (10 of 12) horses, and 100% of pigs, rabbits, hamsters and rats. Notably, 36.4% of all S. aureus identified were MRSA. Methicillin-resistant S. aureus was present in clinical samples from 12 of 487 dogs (2.5%), 6 of 48 cats (12.5%), 5 of 12 horses (42%), and 1 of 2 pigs (50%). The 24 MRSA isolates resolved into 4 PFGE clones: USA100 (50%), USA300 (16.7%), USA500 (20.8%) and USA800 (12.5%) and 6 sequence types (ST5, ST8, ST105, ST830, and ST986) or 2 clonal complexes, CC5 and CC8. Five major virulence profiles (clusters A to E) were observed in these MRSA isolates. Genotypic and virulence profiles of cats and dogs were more similar to each other than to those of horses. A Panton-Valentine leukocidin positive isolate with ST8:USA300 background was identified in a pig causing skin and soft infection. Conclusion: The presence of human MRSA clones in these animals suggests possible reverse zoonotic transmission. This study reports the first case of a USA300 genotype in a pig. Presence of multiple virulence profiles within a MRSA genotype in these animals suggests the potential of emergence of new MRSA clones by gaining or losing additional virulence genes.

Emergence and Spread of Community-Associated Methicillin-Resistant Staphylococcus aureus in Rural Wisconsin, 1989 to 1999

ABSTRACT We investigated the emergence and spread of community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) in central and northern Wisconsin by determining the temporal and clonal relationships and geographic expansion among 581 of 956 clinical isolates of MRSA collected between 1989 and 1999. Based on EcoRI plasmid profiles (PP), two types, PP-11 and PP-13, were highly stable over time and were consistently associated with multidrug-sensitive strains recovered from outpatients treated at Native American community clinics. Pulsed-field gel electrophoresis (PFGE) yielded six major clonal groups (MCGs) and 19 minor clonal groups. The six MCGs represented 82% of the isolates. All strains with either PP-11 or -13 were present in MCG-2. Eighty-nine percent of the isolates in MCG-2 originated from Native American clinics, and 90% belonged to two PFGE types (19 and 20), the types associated with an outbreak of MRSA in a Native American community in 1992. MCG-2 isolates were multidrug sensitive, harbored type IVa staphylococcal cassette chromosome mec, and were very closely related by PFGE to the Midwestern CA-MRSA strain MW2. MCG-2 strains were mostly obtained from skin infections and affected patients with a mean age of 24 (±18.0) years. MCG-2 strains spread to four additional Native American communities and 20 other communities. Our findings suggest that CA-MRSA in Wisconsin likely originated in Native American communities in the early 1990s and since has become widespread throughout the state. Two early CA-MRSA strains (WI-33 and WI-34) in Wisconsin represent progenitors of the MW2 strain, based on their almost indistinguishable genotypic characteristics.

Leptotrichia amnionii sp. nov., a Novel Bacterium Isolated from the Amniotic Fluid of a Woman after Intrauterine Fetal Demise

ABSTRACT A novel bacterium was isolated and characterized from the amniotic fluid of a woman who experienced intrauterine fetal demise in the second trimester of pregnancy. The bacterium was a slow-growing, gram-negative anaerobic coccobacillus belonging to the genus Leptotrichia. Unlike Leptotrichia sanguinegens, the isolate did not grow in chopped-meat glucose broth or on sheep blood agar upon subculturing. The isolate was characterized by sequencing and analyzing its 16S rRNA gene. The 1,493-bp 16S ribosomal DNA sequence had only 96% homology with L. sanguinegens. Several phylogenetic analyses indicated that L. amnionii is a distinct species and most closely related to L. sanguiegens.

Virulence Genes and Genotypic Associations in Nasal Carriage, Community-Associated Methicillin-Susceptible and Methicillin-Resistant USA400 Staphylococcus aureus Isolates

ABSTRACT It is not well understood why strains of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), a major cause of skin and soft tissue infections, became successful so quickly, overtaking the place of methicillin-sensitive S. aureus (MSSA) in many communities. To evaluate the genetic basis of differences in their virulence traits, 293 S. aureus isolates consisting of three cohorts, genotypically defined clinical CA-MRSA (n = 77), clinical MSSA (n = 103), and nasal carriage MSSA (n = 113), collected over a 19-year period in two Midwestern states in the United States, were (i) extensively genotyped and (ii) screened for 40 known virulence genes which included those for enterotoxins, leukocidins, hemolysins, and surface proteins and several newly identified putative toxin genes from the USA400 lineage of CA-MRSA. Genotypically, nasal carriage and clinical MSSA isolates were much more diverse than was the CA-MRSA group, which was found to be of USA400 lineage only. Virulence gene profiles of the three groups showed that CA-MRSA strains harbored significantly higher percentages (≥95%; P value, <0.05) of the sea, sec, sec4, seg2, seh, sek, sel, sel2, ear, ssl1, lpl10, lukSF-PV, lukD, lukE, and clfA genes than did the carriage and the clinical MSSA group (range, 0% to 58%). Genes of the enterotoxin gene cluster, seg, sei, sem, sen, and seo, were present in the clinical and carriage isolates but not in the CA-MRSA group. These results suggest that the presence of additional virulence factors in USA400 CA-MRSA strains compared to the nasal carriage and clinical MSSA strains probably contributed to their enhanced virulence.

Fatal Brain Abscess due to Community-Associated Methicillin-Resistant Staphylococcus aureus Strain USA300

We report a fatal case of brain abscess caused by infection due to a community-associated methicillin-resistant Staphylococcus aureus strain (USA300) in a 37-year-old incarcerated woman with a history of furunculosis and injection drug use. Community-onset pyogenic brain abscess should be added to the growing list of life-threatening invasive infections caused by epidemic community-acquired methicillin-resistant S. aureus.

An analysis of PAX1 in the development of vertebral malformations.

Due to the sporadic occurrence of congenital vertebral malformations, traditional linkage approaches to identify genes associated with human vertebral development are not possible. We therefore identified PAX1 as a candidate gene in vertebral malformations and congenital scoliosis due to its mutation in the undulated mouse. We performed DNA sequence analysis of the PAX1 gene in a series of 48 patients with congenital vertebral malformations, collectively spanning the entire vertebral column length. DNA sequence coding variants were identified in the heterozygous state in exon 4 in two male patients with thoracic vertebral malformations. One patient had T9 hypoplasia, T12 hemivertebrae and absent T10 pedicle, incomplete fusion of T7 posterior elements, ventricular septal defect, and polydactyly. This patient had a CCC (Pro)→CTC (Leu) change at amino acid 410. This variant was not observed in 180 chromosomes tested in the National Institute of Environmental Health Sciences (NIEHS) single nucleotide polymorphism (SNP) database and occurred at a frequency of 0.3% in a diversity panel of 1066 human samples. The second patient had a T11 wedge vertebra and a missense mutation at amino acid 413 corresponding to CCA (Pro)→CTA (Leu). This particular variant has been reported to occur in one of 164 chromosomes in the NIEHS SNP database and was found to occur with a similar frequency of 0.8% in a diversity panel of 1066 human samples. Although each patients mother was clinically asymptomatic and heterozygous for the respective variant allele, the possibility that these sequence variants have clinical significance is not excluded.