The limitations of in vitro experimentation in understanding biofilms and chronic infection

We have become increasingly aware that during infection, pathogenic bacteria often grow in multi- cellular biofilms which are often highly resistant to antibacterial strategies. In order to understand how biofilms form and contribute to infection, in vitro biofilm systems such as microtitre plate as- says and flow cells, have been heavily used by many research groups around the world. Whilst these methods have greatly increased our understanding of the biology of biofilms, it is becoming increasingly apparent that many of our in vitro methods do not accurately represent in vivo conditions. Here we present a systematic review of the most widely used in vitro biofilm systems, and we discuss why they are not always representative of the in vivo biofilms found in chronic infections. We present examples of methods that will help us to bridge the gap between in vitro and in vivo biofilm work, so that our bench-side data can ultimately be used to improve bedside treatment

The prevalence, antibiotic resistance and mecA characterization of coagulase negative staphylococci recovered from non-healthcare settings in London, UK

Background Coagulase negative staphylococci (CoNS) are important reservoirs of antibiotic resistance genes and associated mobile genetic elements and are believed to contribute to the emergence of successful methicillin resistant Staphylococcus aureus (MRSA) clones. Although, these bacteria have been linked to various ecological niches, little is known about the dissemination and genetic diversity of antibiotic resistant CoNS in general public settings. Methods Four hundred seventy-nine samples were collected from different non-healthcare/general public settings in various locations (n = 355) and from the hands of volunteers (n = 124) in London UK between April 2013 and Nov 2014. Results Six hundred forty-three staphylococcal isolates belonging to 19 staphylococcal species were identified. Five hundred seventy-two (94%) isolates were resistant to at least one antibiotic, and only 34 isolates were fully susceptible. Sixty-eight (11%) mecA positive staphylococcal isolates were determined in this study. SCCmec types were fully determined for forty-six isolates. Thirteen staphylococci (19%) carried SCCmec V, followed by 8 isolates carrying SCCmec type I (2%), 5 SCCmec type IV (7%), 4 SCCmec type II (6%), 1 SCCmec type III (2%), 1 SCCmec type VI (2%), and 1 SCCmec type VIII (2%). In addition, three isolates harboured a new SCCmec type 1A, which carried combination of class A mec complex and ccr type 1. MLST typing revealed that all S. epidermidis strains possess new MLST types and were assigned the following new sequence types: ST599, ST600, ST600, ST600, ST601, ST602, ST602, ST603, ST604, ST605, ST606, ST607 and ST608. Conclusions The prevalence of antibiotic resistant staphylococci in general public settings demonstrates that antibiotics in the natural environments contribute to the selection of antibiotic resistant microorganisms. The finding of various SCCmec types in non-healthcare associated environments indicates the complexity of SCCmec. We also report on new MLST types that were assigned for all S. epidermidis isolates, which demonstrates the genetic variability of these isolates

Prevalence and molecular characteristics of Methicillin-Resistant Staphylococcus aureus (MRSA) in organic pig herds in The Netherlands

The prevalence of the methicillin-resistant Staphylococcus aureus (MRSA) among conventional pig herds in the Netherlands is high (around 71%). Nevertheless, information about the prevalence of MRSA among organic pig herds is lacking. Here, we report a study on 24 of the 49 organic pig herds in the Netherlands. The prevalence of MRSA positive herds showed to be 21%. The genetic characteristics of the MRSA isolates were similar to MRSA CC398 described in conventional pigs except one exceptional HA-MRSA CC30 found in one herd, which was presumably caused by human to animal transmission. This resulted in a prevalence of MRSA CC398 in the organic herds of 16.7%

Methicillin-resistant coagulase-negative staphylococci on pig farms as a reservoir of heterogeneous staphylococcal cassette chromosome mec elements

Methicillin-resistant Staphylococcus aureus (MRSA) likely originated by acquisition of the staphylococcal cassette chromosome mec (SCCmec) from coagulase-negative staphylococci (CNS). However, it is unknown whether the same SCCmec types are present in MRSA and CNS that reside in the same niche. Here we describe a study to determine the presence of a potential mecA reservoir among CNS recovered from 10 pig farms. The 44 strains belonged to 10 different Staphylococcus species. All S. aureus strains belonged to sequence type 398 (ST398), with SCCmec types V and IVa. Type IVc, as well as types III and VI, novel subtypes of type IV, and not-typeable types, were found in CNS. S. aureus, S. epidermidis, and S. haemolyticus shared SCCmec type V. The presence of SCCmec type IVc in several staphylococcal species isolated from one pig farm is noteworthy, suggesting exchange of this SCCmec type in CNS, but the general distribution of this SCCmec type still has to be established. In conclusion, this study shows that SCCmec types among staphylococcal species on pig farms are heterogeneous. On two farms, more than one recovered staphylococcal species harbored the same SCCmec type. We conclude that staphylococci on pig farms act as a reservoir of heterogeneous SCCmec elements. These staphylococci may act as a source for transfer of SCCmec to S. aureus

Prevalence and molecular characteristics of Methicillin-Resistant Staphylococcus aureus (MRSA) in organic pig herds in The Netherlands

The prevalence of the methicillin-resistant Staphylococcus aureus (MRSA) among conventional pig herds in the Netherlands is high (around 71%). Nevertheless, information about the prevalence of MRSA among organic pig herds is lacking. Here, we report a study on 24 of the 49 organic pig herds in the Netherlands. The prevalence of MRSA positive herds showed to be 21%. The genetic characteristics of the MRSA isolates were similar to MRSA CC398 described in conventional pigs except one exceptional HA-MRSA CC30 found in one herd, which was presumably caused by human to animal transmission. This resulted in a prevalence of MRSA CC398 in the organic herds of 16.7%

Methicillin-resistant coagulase-negative staphylococci on pig farms as a reservoir of heterogeneous staphylococcal cassette chromosome mec elements

Methicillin-resistant Staphylococcus aureus (MRSA) likely originated by acquisition of the staphylococcal cassette chromosome mec (SCCmec) from coagulase-negative staphylococci (CNS). However, it is unknown whether the same SCCmec types are present in MRSA and CNS that reside in the same niche. Here we describe a study to determine the presence of a potential mecA reservoir among CNS recovered from 10 pig farms. The 44 strains belonged to 10 different Staphylococcus species. All S. aureus strains belonged to sequence type 398 (ST398), with SCCmec types V and IVa. Type IVc, as well as types III and VI, novel subtypes of type IV, and not-typeable types, were found in CNS. S. aureus, S. epidermidis, and S. haemolyticus shared SCCmec type V. The presence of SCCmec type IVc in several staphylococcal species isolated from one pig farm is noteworthy, suggesting exchange of this SCCmec type in CNS, but the general distribution of this SCCmec type still has to be established. In conclusion, this study shows that SCCmec types among staphylococcal species on pig farms are heterogeneous. On two farms, more than one recovered staphylococcal species harbored the same SCCmec type. We conclude that staphylococci on pig farms act as a reservoir of heterogeneous SCCmec elements. These staphylococci may act as a source for transfer of SCCmec to S. aureus

Staphylococcus aureus ST398 gene expression profiling during ex vivo colonization of porcine nasal epithelium

Background Staphylococcus aureus is a common human and animal opportunistic pathogen. In humans nasal carriage of S. aureus is a risk factor for various infections. Methicillin-resistant S. aureus ST398 is highly prevalent in pigs in Europe and North America. The mechanism of successful pig colonization by MRSA ST398 is poorly understood. Previously, we developed a nasal colonization model of porcine nasal mucosa explants to identify molecular traits involved in nasal MRSA colonization of pigs. Results We report the analysis of changes in the transcription of MRSA ST398 strain S0462 during colonization on the explant epithelium. Major regulated genes were encoding metabolic processes and regulation of these genes may represent metabolic adaptation to nasal mucosa explants. Colonization was not accompanied by significant changes in transcripts of the main virulence associated genes or known human colonization factors. Here, we documented regulation of two genes which have potential influence on S. aureus colonization; cysteine extracellular proteinase (scpA) and von Willebrand factor-binding protein (vWbp, encoded on SaPIbov5). Colonization with isogenic-deletion strains (?vwbp and ?scpA) did not alter the ex vivo nasal S. aureus colonization compared to wild type. Conclusions Our results suggest that nasal colonization with MRSA ST398 is a complex event that is accompanied with changes in bacterial gene expression regulation and metabolic adaptation.BN/BionanoscienceApplied Science

Evaluation of porcine mucosa explants after cultivation by means of light microscopy.

<p>Sections of 4 µm thickness were stained by immunohistochemistry to evaluate the apoptosis of cells. TUNEL-positive cells in the epithelium are indicated with white arrows (panel A). Panel B shows the thickness of the epithelium after staining with haematoxylin-eosin (indicated with a white arrow).</p

Scanning electron micrographs of porcine nasal epithelium.

<p>Epithelial cells at 0 h (A) and after 72 h (B) of <i>ex vivo</i> cultivation.</p