Antibiotic resistance and phylogenetic characterization of Acinetobacter baumannii strains isolated from commercial raw meat in Switzerland.

The spread of antibiotic-resistant bacteria through food has become a major public health concern because some important human pathogens may be transferred via the food chain. Acinetobacter baumannii is one of the most life-threatening gram-negative pathogens; multidrug-resistant (MDR) clones of A. baumannii are spreading worldwide, causing outbreaks in hospitals. However, the role of raw meat as a reservoir of A. baumannii remains unexplored. In this study, we describe for the first time the antibiotic susceptibility and fingerprint (repetitive extragenic palindromic PCR [rep-PCR] profile and sequence types [STs]) of A. baumannii strains found in raw meat retailed in Switzerland. Our results indicate that A. baumannii was present in 62 (25.0%) of 248 (CI 95%: 19.7 to 30.9%) meat samples analyzed between November 2012 and May 2013, with those derived from poultry being the most contaminated (48.0% [CI 95%: 37.8 to 58.3%]). Thirty-nine strains were further tested for antibiotic susceptibility and clonality. Strains were frequently not susceptible (intermediate and/or resistant) to third- and fourth-generation cephalosporins for human use (i.e., ceftriaxone [65%], cefotaxime [32%], ceftazidime [5%], and cefepime [2.5%]). Resistance to piperacillin-tazobactam, ciprofloxacin, colistin, and tetracycline was sporadically observed (2.5, 2.5, 5, and 5%, respectively), whereas resistance to carbapenems was not found. The strains were genetically very diverse from each other and belonged to 29 different STs, forming 12 singletons and 6 clonal complexes (CCs), of which 3 were new (CC277, CC360, and CC347). RepPCR analysis further distinguished some strains of the same ST. Moreover, some A. baumannii strains from meat belonged to the clonal complexes CC32 and CC79, similar to the MDR isolates responsible for human infections. In conclusion, our findings suggest that raw meat represents a reservoir of MDR A. baumannii and may serve as a vector for the spread of these pathogens into both community and hospital settings.

International Clostridium difficile animal strain collection and large diversity of animal associated strains

BACKGROUND Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory. RESULTS Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates; 4 hosts), 014/020 (11.6%; 8 hosts); 002 (5.4%; 4 hosts) and 012 (5.4%; 5 hosts). Two animal hosts were best represented; cattle with 31 isolates (20 PCR ribotypes; 7 countries) and pigs with 31 isolates (16 PCR ribotypes; 10 countries). CONCLUSIONS This results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen.

Rapid and accurate identification of Escherichia coli K-12 strains

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.

Complete Genome Sequences of Virulent Mycoplasma capricolum subsp. capripneumoniae Strains F38 and ILRI181

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae is a severe epidemic affecting mainly domestic Caprinae species but also affects wild Caprinae species. M. capricolum subsp. capripneumoniae belongs to the "Mycoplasma mycoides cluster." The disease features prominently in East Africa, in particular Kenya, Tanzania, and Ethiopia. CCPP also endangers wildlife and thus affects not only basic nutritional resources of large populations but also expensively built-up game resorts in affected countries. Here, we report the complete sequences of two M. capricolum subsp. capripneumoniae strains: the type strain F38 and strain ILRI181 isolated druing a recent outbreak in Kenya. Both genomes have a G+C content of 24% with sizes of 1,016,760 bp and 1,017,183 bp for strains F38 and ILRI181, respectively.

Grazing behaviour, physical activity and metabolic profile of two Holstein strains in an organic grazing system

The challenge for sustainable organic dairy farming is identification of cows that are well adapted to forage-based production systems. Therefore, the aim of this study was to compare the grazing behaviour, physical activity and metabolic profile of two different Holstein strains kept in an organic grazing system without concentrate supplementation. Twelve Swiss (HCH ; 566 kg body weight (BW) and 12 New Zealand Holstein-Friesian (HNZ ; 530 kg BW) cows in mid-lactation were kept in a rotational grazing system. After an adaptation period, the milk yield, nutrient intake, physical activity and grazing behaviour were recorded for each cow for 7 days. On three consecutive days, blood was sampled at 07:00, 12:00 and 17:00 h from each cow by jugular vein puncture. Data were analysed using linear mixed models. No differences were found in milk yield, but milk fat (3.69 vs. 4.05%, P = 0.05) and milk protein percentage (2.92 vs. 3.20%, P < 0.01) were lower in HCH than in HNZ cows. Herbage intake did not differ between strains, but organic matter digestibility was greater (P = 0.01) in HCH compared to HNZ cows. The HCH cows spent less (P = 0.04) time ruminating (439 vs. 469 min/day) and had a lower (P = 0.02) number of ruminating boli when compared to the HNZ cows. The time spent eating and physical activity did not differ between strains. Concentrations of IGF-1 and T3 were lower (P ≤ 0.05) in HCH than HNZ cows. In conclusion, HCH cows were not able to increase dry matter intake in order to express their full genetic potential for milk production when kept in an organic grazing system without concentrate supplementation. On the other hand, HNZ cows seem to compensate for the reduced nutrient availability better than HCH cows but could not use that advantage for increased production efficiency

The energy expenditure of 2 Holstein cow strains in an organic grazing system

Until recently, measurements of energy expenditure (EE; herein defined as heat production) in respiration chambers did not account for the extra energy requirements of grazing dairy cows on pasture. As energy is first limiting in most pasture-based milk production systems, its efficient use is important. Therefore, the aim of the present study was to compare EE, which can be affected by differences in body weight (BW), body composition, grazing behavior, physical activity, and milk production level, in 2 Holstein cow strains. Twelve Swiss Holstein-Friesian (HCH; 616 kg of BW) and 12 New Zealand Holstein-Friesian (HNZ; 570 kg of BW) cows in the third stage of lactation were paired according to their stage of lactation and kept in a rotational, full-time grazing system without concentrate supplementation. After adaption, the daily milk yield, grass intake using the alkane double-indicator technique, nutrient digestibility, physical activity, and grazing behavior recorded by an automatic jaw movement recorder were investigated over 7d. Using the (13)C bicarbonate dilution technique in combination with an automatic blood sampling system, EE based on measured carbon dioxide production was determined in 1 cow pair per day between 0800 to 1400 h. The HCH were heavier and had a lower body condition score compared with HNZ, but the difference in BW was smaller compared with former studies. Milk production, grass intake, and nutrient digestibility did not differ between the 2 cow strains, but HCH grazed for a longer time during the 6-h measurement period and performed more grazing mastication compared with the HNZ. No difference was found between the 2 cow strains with regard to EE (291 ± 15.6 kJ) per kilogram of metabolic BW, mainly due to a high between-animal variation in EE. As efficiency and energy use are important in sustainable, pasture-based, organic milk production systems, the determining factors for EE, such as methodology, genetics, physical activity, grazing behavior, and pasture quality, should be investigated and quantified in more detail in future studies.

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.

Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. Results Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. Conclusions In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.

High quality draft genomes of the Mycoplasma mycoides subsp. mycoides challenge strains Afadé and B237.

Members of the Mycoplasma mycoides cluster' represent important livestock pathogens worldwide. Mycoplasma mycoides subsp. mycoides is the etiologic agent of contagious bovine pleuropneumonia (CBPP), which is still endemic in many parts of Africa. We report the genome sequences and annotation of two frequently used challenge strains of Mycoplasma mycoides subsp. mycoides, Afadé and B237. The information provided will enable downstream 'omics' applications such as proteomics, transcriptomics and reverse vaccinology approaches. Despite the absence of Mycoplasma pneumoniae like cyto-adhesion encoding genes, the two strains showed the presence of protrusions. This phenotype is likely encoded by another set of genes.

Identifying the roles of the Escherichia coli FtsZ-associated proteins by minimizing the requirements for division

Formation of the FtsZ ring (Z ring) in Escherichia coli is the first step in assembly of the divisome, a molecular machine composed of 14 known proteins which are all required for cell division. Although the biochemical functions of most divisome proteins are unknown, several of these have overlapping roles in ensuring that the Z ring assembles at the cytoplasmic membrane and is active. ^ We identified a single amino acid change in FtsA, R286W, renamed FtsA*, that completely bypasses the requirement for ZipA in cell division. This and other data suggest that FtsA* is a hyperactive form of FtsA that can replace the multiple functions normally assumed by ZipA, which include stabilization of Z rings, recruitment of downstream cell division proteins, and anchoring the Z ring to the membrane. This is the first example of complete functional replacement of an essential prokaryotic cell division protein by another. ^ Cells expressing ftsA* with a complete deletion of ftsK are viable and divide, although many of these ftsK null cells formed multiseptate chains, suggesting a role in cell separation for FtsK. In addition, strains expressing extra ftsAZ, ftsQ, ftsB, zipA or ftsN, were also able to survive and divide in the absence of ftsK. The cytoplasmic and transmembrane domains of FtsQ were sufficient to allow viability and septum formation to ftsK deleted strains. These findings suggest that FtsK is normally involved in stabilizing the divisome and shares functional overlap with other cell division proteins. ^ As well as permitting the removal of other divisome components, the presence of FtsA* in otherwise wild-type cells accelerated Z-ring assembly, which resulted in a significant decrease in the average length of cells. In support of its role in Z-ring stability, FtsA* suppressed the cell division inhibition caused by overexpressing FtsZ. FtsA* did not affect FtsZ turnover within the Z ring as measured by fluorescence recovery after photobleaching. Turnover of FtsA* in the ring was somewhat faster than wild-type FtsA. Yeast two-hybrid data suggest that FtsA* has an increased affinity for FtsZ relative to wild-type FtsA. These results indicate that FtsA* interacts with FtsZ more strongly, and its enhancement of Z ring assembly may explain why FtsA* can permit survival of cells lacking ZipA or FtsK.^

(Table 1) Characteristics of Klebsormidium (filamentous green algae) strains from the Antarctic, Arctic and Slovakia

The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aero-terrestrial, terrestrial, and hydro-terrestrial) from distinct geographical regions (Antarctic - South Shetlands, King George Island, Arctic - Ellesmere Island, Svalbard, Central Europe - Slovakia) were studied. Each strain was exposed to several freezing (-4°C, -40°C, -196°C) and desiccation (+4°C and +20°C) regimes, simulating both natural and semi-natural freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity) was evaluated by chlorophyll a content, viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation injuries. Freezing down to -196°C was the most harmful regime for all studied strains. Freezing at -4°C did not influence the survival of studied strains. Further, freezing down to -40°C (at a speed of 4°C/min) was not fatal for most of the strains. RDA analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4°C; however, freezing at -40°C, as well as desiccation at +20 °C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic, and Central Europe (Slovakia) survived desiccation at temperatures of +4°C, and freezing down to -40°C. It appears that species of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability prevail, are well adapted to freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor is the resilience only found in polar strains as it is also a feature of temperate strains.