Effect of systemic tetracycline on the degradation of tetracycline-impregnated bilayered collagen membranes: an animal study

BACKGROUND: Premature collagen membrane degradation may compromise the outcome of osseous regenerative procedures. Tetracyclines (TTCs) inhibit the catalytic activities of human metalloproteinases. Preprocedural immersion of collagen membranes in TTC and systemic administration of TTC may be possible alternatives to reduce the biodegradation of native collagen membranes. AIM: To evaluate the in vivo degradation of collagen membranes treated by combined TTC immersion and systemic administration. MATERIALS AND METHODS: Seventy-eight bilayered porcine collagen membrane disks were divided into three groups and were immersed in 0, 50, or 100 mg/mL TTC solution. Three disks, one of each of the three groups, were implanted on the calvaria of each of 26 Wistar rats. Thirteen (study group) were administered with systemic TTC (10 mg/kg), while the remaining 13 received saline injections (control group). Calvarial tissues were retrieved after 3 weeks, and histological sections were analyzed by image analysis software. RESULTS: Percentage of remaining collagen area within nonimpregnated membranes was 52.26 ± 20.67% in the study group, and 32.74 ± 13.81% in the control group. Immersion of membranes in 100 mg/mL TTC increased the amount of residual collagen to 63.46 ± 18.19% and 42.82 ± 12.99% (study and control groups, respectively). Immersion in 50 mg/mL TTC yielded maximal residual collagen values: 80.75 ± 14.86% and 59.15 ± 8.01% (study and control groups, respectively). Differences between the TTC concentrations, and between the control and the study groups were statistically significant. CONCLUSIONS: Immersion of collagen membranes in TTC solution prior to their implantation and systemic administration of TTC significantly decreased the membranes' degradation.

Tetracycline regulator expression alters the transcriptional program of mammalian cells

Tetracycline regulated ectopic gene expression is a widely used tool to study gene function. However, the tetracycline regulator (tetR) itself has been reported to cause certain phenotypic changes in mammalian cells. We, therefore, asked whether human myeloid U937 cells expressing the tetR in an autoregulated manner would exhibit alterations in gene expression upon removal of tetracycline.

Efficiency of risk-based vs . random sampling for the monitoring of tetracycline residues in slaughtered calves in Switzerland

In all European Union countries, chemical residues are required to be routinely monitored in meat. Good farming and veterinary practice can prevent the contamination of meat with pharmaceutical substances, resulting in a low detection of drug residues through random sampling. An alternative approach is to target-monitor farms suspected of treating their animals with antimicrobials. The objective of this project was to assess, using a stochastic model, the efficiency of these two sampling strategies. The model integrated data on Swiss livestock as well as expert opinion and results from studies conducted in Switzerland. Risk-based sampling showed an increase in detection efficiency of up to 100% depending on the prevalence of contaminated herds. Sensitivity analysis of this model showed the importance of the accuracy of prior assumptions for conducting risk-based sampling. The resources gained by changing from random to risk-based sampling should be transferred to improving the quality of prior information.

Camel Streptococcus agalactiae populations are associated with specific disease complexes and acquired the tetracycline resistance gene tetM via a Tn916-like element

Camels are the most valuable livestock species in the Horn of Africa and play a pivotal role in the nutritional sustainability for millions of people. Their health status is therefore of utmost importance for the people living in this region. Streptococcus agalactiae, a Group B Streptococcus (GBS), is an important camel pathogen. Here we present the first epidemiological study based on genetic and phenotypic data from African camel derived GBS. Ninety-two GBS were characterized using multilocus sequence typing (MLST), capsular polysaccharide typing and in vitro antimicrobial susceptibility testing. We analysed the GBS using Bayesian linkage, phylogenetic and minimum spanning tree analyses and compared them with human GBS from East Africa in order to investigate the level of genetic exchange between GBS populations in the region. Camel GBS sequence types (STs) were distinct from other STs reported so far. We mapped specific STs and capsular types to major disease complexes caused by GBS. Widespread resistance (34%) to tetracycline was associated with acquisition of the tetM gene that is carried on a Tn916-like element, and observed primarily among GBS isolated from mastitis. The presence of tetM within different MLST clades suggests acquisition on multiple occasions. Wound infections and mastitis in camels associated with GBS are widespread and should ideally be treated with antimicrobials other than tetracycline in East Africa.

Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems

In the present study, the tetracycline-off and Cre/loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP-flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

TET inducible expression of the α4β7-integrin ligand MAdCAM-1 on the blood-brain barrier does not influence the immunopathogenesis of experimental autoimmune encephalomyelitis

Inhibiting the α4 subunit of the integrin heterodimers α4β1 and α4β7 with the mab natalizumab is an effective treatment of multiple sclerosis (MS). Which of the two α4 heterodimers is involved in disease pathogenesis has, however, remained controversial. Whereas the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is ameliorated in β7-integrin-deficient C57BL/6 mice, neutralizing antibodies against the β7-integrin subunit or the α4β7-integrin heterodimer fail to interfere with EAE pathogenesis in the SJL mouse. To facilitate α4β7-integrin-mediated immune-cell trafficking across the blood-brain barrier (BBB), we established transgenic C57BL/6 mice with endothelial cell-specific, inducible expression of the α4β7-integrin ligand mucosal addressin cell adhesion molecule (MAdCAM)-1 using the tetracycline (TET)-OFF system. Although TET-regulated MAdCAM-1 induced α4β7-integrin mediated interaction of α4β7(+) /α4β1(-) T cells with the BBB in vitro and in vivo, it failed to influence EAE pathogenesis in C57BL/6 mice. TET-regulated MAdCAM-1 on the BBB neither changed the localization of central nervous system (CNS) perivascular inflammatory cuffs nor did it enhance the percentage of α4β7-integrin(+) inflammatory cells within the CNS during EAE. In conclusion, our study demonstrates that ectopic expression of MAdCAM-1 at the BBB does not increase α4β7-integrin-mediated immune cell trafficking into the CNS during MOG(aa35-55)-induced EAE.

Evolution of multidrug-resistant Staphylococcus aureus infections in horses and colonized personnel in an equine clinic between 2005 and 2010

A total of 70 Staphylococcus aureus isolates from postoperative infections in hospitalized horses were isolated between January 2005 and January 2011. Among them, 12 isolates were methicillin-susceptible S. aureus (MSSA), 18 were borderline-oxacillin-resistant S. aureus (BORSA), and 40 were methicillin-resistant S. aureus (MRSA). During the same period, the equine clinic personnel were screened for nasal carriage of BORSA and MRSA. Genotyping revealed that BORSA ST1(MLST)-t2863(spa) isolates were responsible for most equine infections and were the main isolates found in colonized members of the personnel between 2005 and 2007, and that in 2007, MRSA ST398-t011-IVa(SCCmec) emerged in infection sites and personnel, replacing BORSA. Besides decreased susceptibility to oxacillin, all MRSA and BORSA of these two major clonal lineages displayed resistance to gentamicin and kanamycin conferred by the aac(6')-Ie-aph(2')-Ia gene and to trimethoprim conferred by dfr(K) in MRSA and dfr(A) in BORSA. All MRSA had additional resistance to tetracycline conferred by tet(M), whereas BORSA generally also display resistance to streptomycin conferred by str. The number of hospital-acquired MRSA infections in horses could be limited after the introduction of basic hygiene measures and personnel decolonization. Two MRSA carriers could not be decolonized using mupirocin, and a year after decolonization, additional members were recolonized with MRSA. Hygiene measures should, therefore, be maintained to limit the transmission of S. aureus between personnel and horses.

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

AIMS: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. METHODS AND RESULTS: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which - in combination with MLST - increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37.5, 33.1 and 8.1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone-sensitive human isolates showed overlapping MLST-flaB types with those of chicken origin, resistant strains showed only 39% of matching types. CONCLUSION: Mainly quinolone-sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.

The increase of methicillin-resistant Staphylococcus aureus (MRSA) and the presence of an unusual sequence type ST49 in slaughter pigs in Switzerland

Background: In years past, methicillin-resistant S. aureus (MRSA) has been frequently detected in pigs in Europe, North America and Asia. Recent, yet sporadic studies have revealed a low occurrence of MRSA in Switzerland. In 2009, a monitoring survey of the prevalence and genetic diversity of methicillin-resistant S. aureus (MRSA) in slaughter pigs in Switzerland was conducted using methods recommended by the EU guidelines, and using a sampling strategy evenly distributed throughout the year and representative of the Swiss slaughter pig population. Monitoring should determine if the overall prevalence of MRSA in the entire country is increasing over the years and if specific multi-resistant MRSA clones are spreading over the country.;Results: In 2009, the nasal cavities of eight out of 405 randomly selected pigs were positive for MRSA, representing a prevalence of 2.0% (95% CI 0.9-3.9). The following year, 23 out of 392 pigs were positive for MRSA [5.9% prevalence (95% CI 3.8-8.7)]. Three multilocus sequence types (ST), four spa types and two types of staphylococcal cassette chromosome mec (SCCmec) elements were detected. The most frequent genotypes were ST398 (MLST)-(spa)t034-V (SCCmec) (n = 18) and ST49-t208-V (n = 7), followed by ST398-t011-V (n = 4), ST398-t1451-V (n = 1), and ST1-t2279-IVc (n = 1). The isolates displayed resistance to beta-lactams [mecA, (31/31); blaZ, (19/31)]; tetracycline [tet(M), (31/31); tet(K), (30/31)] (n = 31); macrolides and lincosamides [erm(C) (4/31) or erm(A) (18/31)] (n = 22); tiamulin [vga(A)v (9/31) or unknown mechanism (18/31)] (n = 27); trimethoprim [dfr(G) (18/31); spectinomycin [ant(9)-Ia (19/31) or unknown mechanism (3/31)] (n = 22); streptomycin [str(19/31)]; sulphamethoxazole (7/31) and ciprofloxacin (n = 1) (mechanisms not determined).;Conclusions: This study is the first to describe the presence of MRSA ST49 in slaughter pigs, and to demonstrate a significant and nearly three-fold increase of MRSA prevalence in pigs within two years. The presence of a specific clonal lineage of MRSA from Switzerland suggests that it has been selected in Swiss pig husbandry. Effective hygiene measures should be enhanced within the entire pig production chain to suppress the spread of these pathogens into the community.

Two novel antibiotic resistance genes, tet(44) and ant(6)-Ib, are located within a transferable pathogenicity island in Campylobacter fetus subsp. fetus

New tetracycline and streptomycin resistance genes, tet(44) and ant(6)-Ib, were identified in Campylobacter fetus subsp. fetus within a transferable pathogenicity island that is typically unique to Campylobacter fetus subsp. venerealis. The 640-amino-acid tetracycline resistance determinant, Tet 44, belongs to a class of proteins that confers resistance to tetracycline and minocycline by ribosomal protection. The 286-amino-acid streptomycin resistance determinant, ANT(6)-Ib, belongs to a family of aminoglycoside nucleotidyltransferases. The resistance phenotypes were demonstrated by gene inactivation and expression.

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Antibiotic resistance profile of Staphylococcus rostri, a new species isolated from healthy pigs

Staphylococcus rostri is a newly described Staphylococcus species that is present in the nasal cavity of healthy pigs. Out of the 225 pigs tested at slaughterhouse, 46.7% carried the new species alone and 22% in combination with Staphylococcus aureus. An antibiotic resistance profile was determined for S. rostri and compared to that of S. aureus isolated from the same pig. Resistance to tetracycline specified by tet(M), tet(K) and tet(L), streptomycin (str(pS194)), penicillin (blaZ), trimethoprim (dfr(G)), and erythromycin and clindamycin (erm genes), were found in both species; however, with the exception of streptomycin and trimethoprim, resistance was higher in S. aureus. S. rostri isolates display very low genetic diversity as demonstrated by pulsed-field gel electrophoresis, which generated two major clusters. Several clonal complexes (CC1, CC5, CC9, CC30 and CC398) were identified in S. aureus with CC 9 and CC 398 being the most frequent. Our study gives the first overview of the distribution, genetic relatedness, and resistance profile of one coagulase-negative Staphylococcus species that is commonly present in the nares of healthy pigs in Switzerland, and shows that S. rostri may harbor resistance genes associated with transferable elements like Tn916.

Radial extracorporeal shock wave therapy (rESWT) induces new bone formation in vivo: results of an animal study in rabbits

The aim of this study was to investigate if radial extracorporeal shock wave therapy (rESWT) induces new bone formation and to study the time course of ESWT-induced osteogenesis. A total of 4000 impulses of radial shock waves (0.16 mJ/mm²) were applied to one hind leg of 13 New Zealand white rabbits with the contralateral side used for control. Treatment was repeated after 7 days. Fluorochrome sequence labeling of new bone formation was performed by subcutaneous injection of tetracycline, calcein green, alizarin red and calcein blue. Animals were sacrificed 2 weeks (n = 4), 4 weeks (n = 4) and 6 weeks (n = 5) after the first rESWT and bone sections were analyzed by fluorescence microscopy. Deposits of fluorochromes were classified and analyzed for significance with the Fisher exact test. rESWT significantly increased new bone formation at all time points over the 6-week study period. Intensity of ossification reached a peak after 4 weeks and declined at the end of the study. New bone formation was significantly higher and persisted longer at the ventral cortex, which was located in the direction to the shock wave device, compared with the dorsal cortex, emphasizing the dose-dependent process of ESWT-induced osteogenesis. No traumata, such as hemorrhage, periosteal detachment or microfractures, were observed by histologic and radiologic assessment. This is the first study demonstrating low-energy radial shock waves to induce new bone formation in vivo. Based on our results, repetition of ESWT in 6-week intervals can be recommended. Application to bone regions at increased fracture risk (e.g., in osteoporosis) are possible clinical indications.

Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission

Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to beta-lactams (mecA and blaZ), aminoglycosides [str and aac(6')-Ie-aph(2')-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.