Mary E. Stemper

Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission†

ABSTRACT A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.

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.

AEROMONAS ISOLATES FROM HUMAN DIARRHEIC STOOL AND GROUNDWATER COMPARED BY PULSED - FIELD GEL ELECTROPHORESIS

Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients’ drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas. Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas-positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures.

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.

Shift in Staphylococcus aureus Clone Linked to an Infected Tattoo

A retrospective investigation of skin and soft tissue infections caused by community-associated methicillin-resistant Staphylococcus aureus (MRSA) strains among inmates in a Wisconsin correctional facility suggested a shift in MRSA genotype. Case timeline indicated a displacement of USA400 clone by USA300 clone. The USA300 index case was associated with an infected new tattoo.

Necrotizing Fasciitis Due to a Methicillin-Sensitive Staphylococcus aureus Isolate Harboring an Enterotoxin Gene Cluster

ABSTRACT Benign papular eruption on the left leg of a 72-year-old diabetic man developed into rapidly spreading necrotizing fasciitis despite antimicrobial therapy and surgical debridements. This led to eventual amputation to control the infection. The etiological agent was a Staphylococcus aureus isolate harboring the enterotoxin gene cluster seg, sei, sem, sen, and seo but lacked all common toxin genes, including Panton-Valentine leukocidin.

New classes of Gram-positive selective antibacterials: inhibitors of MRSA and surrogates of the causative agents of anthrax and tuberculosis.

Abstract An antimicrobial phenolic stilbene, (E)-3-hydroxy-5-methoxystilbene, 1 was recently isolated from the leaves of Comptonia peregrina (L.) Coulter and shown to possess inhibitory activity against several Gram-positive bacteria, including isolates of methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium bovis BCG, and avirulent Bacillus anthracis (Sterne strain), among others. These results prompted the design and synthesis of two new classes of compounds, phenoxystyrenes and phenothiostyrenes, as analogs of the natural antimicrobial stilbene. These and additional stilbenoid analogs were synthesized using new, efficient, copper-mediated coupling strategies. Minimum inhibitory concentration (MIC) antimicrobial assays were performed on all compounds prepared. These preliminary structure–activity relationship studies indicated that both new classes of synthetic analogs, as well as the stilbenes, show promising activity against Gram-positive bacteria when at least one phenolic moiety is present, but not when absent. The potencies of the phenolic phenoxystyrenes and phenothiostyrenes were found to be comparable to those of the phenolic stilbenes tested.