Paul D. Fey

Ceftriaxone-Resistant Salmonella Infection Acquired by a Child from Cattle

BACKGROUND The emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. Resistance to ceftriaxone and the fluoroquinolones, which are used to treat invasive salmonella infections, is rare in the United States. We analyzed the molecular characteristics of a ceftriaxone-resistant strain of Salmonella enterica serotype typhimurium isolated from a 12-year-old boy with fever, abdominal pain, and diarrhea. METHODS We used pulsed-field gel electrophoresis and analysis of plasmids and beta-lactamases to compare the ceftriaxone-resistant S. enterica serotype typhimurium from the child with four isolates of this strain obtained from cattle during a local outbreak of salmonellosis. RESULTS The ceftriaxone-resistant isolate from the child was indistinguishable from one of the isolates from cattle, which was also resistant to ceftriaxone. Both ceftriaxone-resistant isolates were resistant to 13 antimicrobial agents; all but one of the resistance determinants were on a conjugative plasmid of 160 kb that encoded the functional group 1 beta-lactamase CMY-2. Both ceftriaxone-resistant isolates were closely related to the three other salmonella isolates obtained from cattle, all of which were susceptible to ceftriaxone. CONCLUSIONS This study provides additional evidence that antibiotic-resistant strains of salmonella in the United States evolve primarily in livestock. Resistance to ceftriaxone, the drug of choice for invasive salmonella disease, is a public health concern, especially with respect to children, since fluoroquinolones, which can also be used to treat this disease, are not approved for use in children.

Management of Adults With Hospital-acquired and Ventilator-associated Pneumonia: 2016 Clinical Practice Guidelines by the Infectious Diseases Society of America and the American Thoracic Society.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. IDSA considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patients individual circumstances.These guidelines are intended for use by healthcare professionals who care for patients at risk for hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP), including specialists in infectious diseases, pulmonary diseases, critical care, and surgeons, anesthesiologists, hospitalists, and any clinicians and healthcare providers caring for hospitalized patients with nosocomial pneumonia. The panels recommendations for the diagnosis and treatment of HAP and VAP are based upon evidence derived from topic-specific systematic literature reviews.

Coagulase-Negative Staphylococcal Infections

Coagulase-negative staphylococci (CNS) are differentiated from the closely related but more virulent Staphylococcus aureus by their inability to produce free coagulase. Currently, there are over 40 recognized species of CNS. These organisms typically reside on healthy human skin and mucus membranes, rarely cause disease, and are most frequently encountered by clinicians as contaminants of microbiological cultures. However, CNS have been increasingly recognized to cause clinically significant infections. The conversion of the CNS from symbiont to human pathogen has been a direct reflection of the use of indwelling medical devices. This article deals with the clinical syndromes, epidemiology, prevention, and management of infections caused by this unique group of organisms.

A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes

ABSTRACT To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen. IMPORTANCE Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website (http://app1.unmc.edu/fgx/) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems. Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website (http://app1.unmc.edu/fgx/) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems.

Extended Spectrum β-Lactamase (ESBL)-Producing Enterobacteriaceae Considerations for Diagnosis, Prevention and Drug Treatment

Extended spectrum beta-lactamase (ESBL)-producing organisms pose unique challenges to clinical microbiologists, clinicians, infection control professionals and antibacterial-discovery scientists. ESBLs are enzymes capable of hydrolysing penicillins, broad-spectrum cephalosporins and monobactams, and are generally derived from TEM and SHV-type enzymes. ESBLs are often located on plasmids that are transferable from strain to strain and between bacterial species. Although the prevalence of ESBLs is not known, it is clearly increasing, and in many parts of the world 10-40% of strains of Escherichia coli and Klebsiella pneumoniae express ESBLs. ESBL-producing Enterobacteriaceae have been responsible for numerous outbreaks of infection throughout the world and pose challenging infection control issues. Clinical outcomes data indicate that ESBLs are clinically significant and, when detected, indicate the need for the use of appropriate antibacterial agents. Unfortunately, the laboratory detection of ESBLs can be complex and, at times, misleading. Antibacterial choice is often complicated by multi-resistance. Many ESBL-producing organisms also express AmpC beta-lactamases and may be co-transferred with plasmids mediating aminoglycoside resistance. In addition, there is an increasing association between ESBL production and fluoroquinolone resistance. Although in in vitro tests ESBLs are inhibited by beta-lactamase inhibitors such as clavulanic acid, the activity of beta-lactam/beta-lactamase inhibitor combination agents is influenced by the bacterial inoculum, dose administration regimen and specific type of ESBL present. Currently, carbapenems are regarded as the drugs of choice for treatment of infections caused by ESBL-producing organisms. Unfortunately, use of carbapenems has been associated with the emergence of carbapenem-resistant bacterial species such as Stenotrophomonas sp. or Pseudomonas sp.Extended spectrum β-lactamase (ESBL)-producing organisms pose unique challenges to clinical microbiologists, clinicians, infection control professionals and antibacterial-discovery scientists. ESBLs are enzymes capable of hydrolysing penicillins, broad-spectrum cephalosporins and monobactams, and are generally derived from TEM and SHV-type enzymes. ESBLs are often located on plasmids that are transferable from strain to strain and between bacterial species. Although the prevalence of ESBLs is not known, it is clearly increasing, and in many parts of the world 10–40% of strains of Escherichia coli and Klebsiella pneumoniae express ESBLs.ESBL-producing Enterobacteriaceae have been responsible for numerous outbreaks of infection throughout the world and pose challenging infection control issues. Clinical outcomes data indicate that ESBLs are clinically significant and, when detected, indicate the need for the use of appropriate antibacterial agents. Unfortunately, the laboratory detection of ESBLs can be complex and, at times, misleading.Antibacterial choice is often complicated by multi-resistance. Many ESBL-producing organisms also express AmpC β-lactamases and may be co-transferred with plasmids mediating aminoglycoside resistance. In addition, there is an increasing association between ESBL production and fluoroquinolone resistance. Although in in vitro tests ESBLs are inhibited by β-lactamase inhibitors such as clavulanic acid, the activity of β-lactam/β-lactamase inhibitor combination agents is influenced by the bacterial inoculum, dose administration regimen and specific type of ESBL present. Currently, carbapenems are regarded as the drugs of choice for treatment of infections caused by ESBL-producing organisms. Unfortunately, use of carbapenems has been associated with the emergence of carbapenem-resistant bacterial species such as Stenotrophomonas sp. or Pseudomonas sp.

Characterization of the Importance of Staphylococcus epidermidis Autolysin and Polysaccharide Intercellular Adhesin in the Pathogenesis of Intravascular Catheter-Associated Infection in a Rat Model

A rat central venous catheter (CVC) infection model was used to assess the importance of the proteinacious autolysin (AtlE) and the polysaccharide intercellular adhesin (PIA) in the pathogenesis of Staphylococcus epidermidis CVC-associated infection. Wild-type (wt) S. epidermidis O-47 was significantly more likely to cause a CVC infection than was either of the isogenic mutant strains (AtlE-negative [O-47mut1] or PIA-negative [O-47mut2]). Bacteria were retrieved from the explanted catheters of 87.5% of rats inoculated with S. epidermidis O-47, compared with 25% of rats challenged with either S. epidermidis O-47mut1 or O-47mut2 (P=.007). Peripheral bacteremia was documented in 75% of rats challenged with S. epidermidis O-47, compared with 12.5% and 25% challenged with O-47mut1 and O-47mut2, respectively (P=.009). Metastatic disease was more common in rats inoculated with wt S. epidermidis, compared with AtlE- or PIA-deficient mutants. These results confirm the importance of initial adherence, associated with AtlE, and biofilm production, mediated by PIA, in the pathogenesis of S. epidermidis experimental CVC infection.

Current concepts in biofilm formation of Staphylococcus epidermidis

Staphylococcus epidermidis is a highly significant nosocomial pathogen mediating infections primarily associated with indwelling biomaterials (e.g., catheters and prostheses). In contrast to Staphylococcus aureus, virulence properties associated with S. epidermidis are few and biofilm formation is the defining virulence factor associated with disease, as demonstrated by animal models of biomaterial-related infections. However, other virulence factors, such as phenol-soluble modulins and poly-gamma-DL-glutamic acid, have been recently recognized that thwart innate immune system mechanisms. Formation of S. epidermidis biofilm is typically considered a four-step process consisting of adherence, accumulation, maturation and dispersal. This article will discuss recent advances in the study of these four steps, including accumulation, which can be either polysaccharide or protein mediated. It is hypothesized that studies focused on understanding the biological function of each step in staphylococcal biofilm formation will yield new treatment modalities to treat these recalcitrant infections.

Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.

Characterization of Plasmids Carrying CMY-2 from Expanded-Spectrum Cephalosporin-Resistant Salmonella Strains Isolated in the United States between 1996 and 1998

ABSTRACT Sequencing of DNA from 15 expanded-spectrum cephalosporin (e.g., ceftriaxone)-resistant Salmonella isolates obtained in the United States revealed that resistance to ceftriaxone in all isolates was mediated by cmy-2. Hybridization patterns revealed three plasmid structures containing cmy-2 in these 15 isolates. These data suggest that the spread of cmy-2 among Salmonella strains is occurring through mobilization of the cmy-2 gene into different plasmid backbones and consequent horizontal transfer by conjugation.

Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis

ABSTRACT The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx 1 and stx 2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.