Detection of Mycobacterium tuberculosis Complex Genetic Material in a Free-Living Brown Bear (Ursus arctos).

Mycobacterium tuberculosis complex (MTBC) has rarely been detected in bears (Ursidae). We describe detection of MTBC genetic material using a single-tube, high-multiplex PCR and fluorescence-based detection system in a throat swab collected from a free-living, problem individual during immobilization and telemetry collar deployment. Mycobacterial culture was negative in all samples.

A New Approach to Imaging and Rapid Microbiome Identification for Prostate Cancer Patients Undergoing Radiotherapy.

(1) Background: Little is known about the impact of urinary microflora, in particular, its effects on side effects after radiotherapy. The use of mass spectrometry identification method (MALDI) may bring a new look at the issue of the composition and significance of the urinary microbiome. This study aimed to use the mass spectrometry identification method (MALDI) to identify the microbiome of urine samples collected from 50 irradiated prostate cancer patients. (2) Methods: Blood and urine samples were collected before gold marker implantation, at the start and last day of radiotherapy, 1, 4 months after. Patients do not always collect the urine from the midstream; therefore, samples were collected from the first void and midstream in 12 patients for MALDI analysis; in the remaining 38 patients-from the midstream void for MALDI and biochemical analysis. (3) Results: Microorganisms were present in 140/181 urine samples. We found 33 different species 3G(-) and 30G(+). The most frequently isolated strains were: Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis, Enterococcus faecalis, and Micrococcus luteus. When comparing the type of urine samples, bacteria were more common in samples from the first-void urine than from the midstream one. The absence of bacteria was found in 12.2% of samples from the first-void urine and in 24.7% from the midstream. There was no difference in the total incidence of species between streams (p = 0.85). Before fiducial implantation, the total number of detected bacterial species was significantly higher in comparison to the end of radiotherapy (p = 0.038), indicating that the administered therapy resulted in depleting the local microbiome. One month after radiotherapy, an increase in the number of isolated bacteria was observed. The number of bacterial species in urine did not correlate with blood parameters. The presence of leukocytes (p = 0.013) and proteins (p = 0.004) in urine was related to a greater variety of bacteria found in urine specimens. (4) Conclusions: We obtained a similar spectrum of bacteria from the initial and middle urine streams. We also showed that there is a change in bacteria species affected by the treatment of prostate cancer patients, with both antibiotics before gold fiducial implantation and radiotherapy.

Mating system and extra-pair paternity in the Fan-tailed Gerygone Gerygone flavolateralis in relation to parasitism by the Shining Bronze-cuckoo Chalcites lucidus.

Extra-pair copulation can increase genetic diversity and offspring fitness. However, it may also increase intra-nest variability in avian hosts of brood parasites, which can decrease the discrimination ability of host parents towards the parasite. In New Caledonia, the Fan-tailed Gerygone (Gerygone flavolateralis), which is parasitized by the Shining Bronze-cuckoo (Chalcites lucidus), has two nestling morphs, dark and bright, that can occur in monomorphic and polymorphic broods. Gerygone parents recognize and eject parasite nestlings from their nest, but the presence of polymorphic broods may increase the chances of recognition errors. Using 17 microsatellite markers, we investigated the mating system of the Fan-tailed Gerygone to understand the mechanisms underlying nestling polymorphism. We hypothesised that extra-pair copulations would lead to a higher proportion of polymorphic broods caused by higher genetic variability, thus creating a trade-off between genetic benefits and host defence reliability. Extra-pair paternity occurred in 6 of 36 broods, which resulted in 6 of 69 offspring sired by extra-pair males. Broods with and without mixed paternity were comparably often parasitized. Extra-pair paternity did not influence the proportions of bright, dark and polymorphic broods. Compared to bright siblings in polymorphic broods, dark nestlings tended to have lower heterozygosity, particularly in loci associated with skin coloration. The results also suggested that there is no obstacle for genetic exchange between individuals from forest and savannah, possibly due to dispersal of offspring. We conclude that the Fan-tailed Gerygone is a socially monogamous species with a low rate of extra-pair paternity compared to closely related species. Extra-pair paternity increased offspring genetic variability without measurable associated costs by brood parasitism. The results highlight the importance of studying host mating systems to assess the trade-offs between host defence and offspring fitness in co-evolutionary arms races.

Effectiveness of different types of hair traps for brown bear research and monitoring.

Non-invasive sampling by hair-trapping is increasingly used worldwide in wildlife research. Despite this rise and the potential of hair samples for ecology and conservation studies, the relative performance of hair collection devices has been rarely tested. Here, we compare the effectiveness of five types of hair traps for brown bears Ursus arctos in the Carpathian Mountains (SE Poland) and test the effects of trap type, season, number of days elapsed since trap installation and trap features on the trapping success in order to provide recommendations for optimal sampling in future studies. The trap types were corral, path-trap, "smola"(beechwood tar) tree-trap, turpentine tree-trap and natural rub. In 2010, we collected 858 hair samples during 2330 inspections of 175 hair traps and found that the most effective traps were smola tree-traps (mean percentage of successful inspections ± SD: 30.2% ± 26.0) and natural rubs (50.8% ± 16.7). Based on this finding, over the following 2 years we focused on 24 smola tree-traps and eight natural rubs. During this long-term survey (2010-2012, 969 inspections, 1322 samples collected) the trapping success increased with time and smola tree-traps achieved similar effectiveness to natural rubs (45.5% ± 29.7 and 45.9 ± 23.4, respectively). We show that when baiting smola tree-traps ten weeks prior to research or monitoring, sampling effectiveness can reach up to 30%. Taking into account the logistical and methodological constraints associated with detecting and using natural rubs for a proper survey design, we recommend using smola tree-traps baited in advance for hair sampling in wildlife studies.

ß-Lactam resistance among Haemophilus influenzae isolates in Poland.

OBJECTIVES: Haemophilus influenzae is a human-specific Gram-negative coccobacillus responsible for a significant number of respiratory tract infections and severe invasive infections such as meningitis and sepsis. The purpose of this study was to characterise the mechanisms of ß-lactam resistance among Polish H. influenzae isolates and to evaluate the resistance detection methods applied. METHODS: This study was conducted on 117 Polish H. influenzae isolates collected in 2012. Minimum inhibitory concentrations were assessed by broth microdilution. All strains were evaluated using the disk diffusion method and the algorithm proposed by the Nordic Committee on Antimicrobial Susceptibility Testing (NordicAST). To detect changes in penicillin-binding protein 3 (PBP3), PCR screening was performed, followed by ftsI gene sequencing. RESULTS: Neither ß-lactamase production nor PBP3 alterations were demonstrated in 76 isolates (65.0%). Susceptibility to ampicillin, amoxicillin, amoxicillin/clavulanic acid, cefuroxime (intravenous) and ceftriaxone was observed in 70.9%, 78.6%, 98.3%, 82.9% and 100% of the isolates, respectively. ß-Lactamase production characterised 21 isolates (17.9%). Screening PCR identified 20 isolates (17.1%) with PBP3 alterations, and according to subsequent ftsI sequencing all these strains were finally recognised as gBLNAR (genetically ß-lactamase-negative, ampicillin-resistant), among which 65.0% were ampicillin-resistant. According to molecular classification of PBP3 alterations, 95.0% of gBLNAR belonged to group II, representing four subgroups IIa-IId. CONCLUSIONS: Haemophilus influenzae resistance to antibiotics requires continuous attention, effective detection methods and a rational policy of antibiotic usage. The algorithm proposed by NordicAST can be applied in routine laboratory work, whereas sequencing of the ftsI gene may be useful in molecular epidemiology studies.

Multiregional dissemination of KPC-producing Klebsiella pneumoniae ST258/ST512 genotypes in Poland, 2010-14.

Objectives: In 2008-09, the KPC carbapenemase epidemiology in Poland was dominated by a Klebsiella pneumoniae ST258 KPC-2 outbreak in Warsaw and its administrative region. The aim of this study was to analyse the situation in 2010-14, with a focus on new outbreaks in other parts of the country. Methods: KPCs were detected in all suspected isolates by PCR. The detailed study was performed on 173 isolates from 2010 to 2012, and included PFGE and MLST, PCR identification of K. pneumoniae clonal group CG258 clades and potential specificity markers ( pilv-1 , IS 66 and prp ), PCR mapping of Tn 4401 transposons, and plasmid analysis by nuclease S1 profiling and PCR-based replicon typing. Results: Six hundred and eight KPC cases were identified in Poland in 2010-14, almost half of which occurred in the Warsaw region, and another half in four other areas. The new outbreaks were caused by four K. pneumoniae CG258 genotypes, different from each other and from the organisms spreading in Warsaw. The new lineages were ST258 or ST512 of clade II, and had specific compositions of potential ST258/ST512 clonal markers. The isolates produced KPC-3 encoded by Tn 4401 a or Tn 4401 b elements on plasmids with single or multiple replicons, including I2, FII K (+/-FIB K ), 'FII Y -like', X3 and R. Of other species, Citrobacter freundii ST17 and Enterobacter cloacae ST254 with KPC-2 were identified in a Warsaw hospital. Conclusions: The study showed remarkable changes in the KPC epidemiology in Poland in 2010-14, which, following the localized regional spread in the early phase, has converted into multiregional dissemination.

KPC-2-producing Klebsiella pneumoniae ST11 in a Children's Hospital in Poland.

Four Klebsiella pneumoniae isolates from children hospitalized over 10 months in an intensive care unit in a children's teaching hospital in Poland were analyzed. All of the isolates belonged to a single pulsotype and sequence type (ST) 11, and produced the KPC-2 carbapenemase and extended-spectrum ß-lactamase (ESBL) CTX-M-15. They were resistant to a variety of antimicrobials, and their ß-lactam resistance patterns were typical for KPC producers. This is one of few cases of identification of KPC (or carbapenemase)-producing K. pneumoniae in a pediatric center in Poland.

Evolution of major histocompatibility complex class I and class II genes in the brown bear.

BACKGROUND: Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. RESULTS: We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. CONCLUSIONS: Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South-north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia.

Molecular characteristics of KPC-producing Enterobacteriaceae at the early stage of their dissemination in Poland, 2008-2009.

After the first report in May 2008, the National Reference Center for Susceptibility Testing confirmed 113 cases of infection or colonization by KPC-producing members of the family Enterobacteriaceae in Poland by the end of 2009. The vast majority of patients were found in 18 hospitals; three patients were diagnosed at outpatient clinics. Most of the institutions were in the Warsaw area, including three hospitals with the highest numbers of cases. When available, the data on previous hospitalizations often indicated that these hospitals were the probable acquisition sites; one patient arrived from New York. The group of 119 unique isolates consisted of Klebsiella pneumoniae (n = 114), followed by Klebsiella oxytoca (n = 3), and Escherichia coli (n = 2). The K. pneumoniae isolates were dominated by the clone sequence type 258 (ST258) (n = 111); others were ST11 and ST23. The ST258 group was heterogeneous, with 28 pulsed-field gel electrophoresis (PFGE) subtypes, ∼25 plasmid profiles, and nine ß-lactamase patterns differing by KPC variants (KPC-2 mainly), and SHV-12, CTX-M-3, and TEM-1-like enzymes. Plasmids carrying bla(KPC) genes varied in size (~48 to 250 kb), structure, and conjugation potential. Transferable IncFII(K) plasmids of ~110 to 160 kb, probably pKpQIL or its derivatives, were observed in all K. pneumoniae clones and in K. oxytoca. Also prevalent were nontypeable pETKp50-like plasmids of ~50 kb, found in K. pneumoniae ST258 and E. coli isolates (ST93 and ST224). Two K. pneumoniae-E. coli pairs from single patients might represent the in vivo transfer of such plasmids. The striking diversity of KPC producers at the early stage of dissemination could result from several introductions of these bacteria into the country, their multidirectional evolution during clonal spread, and transfer of the plasmids.

The current status of invasive pneumococcal disease in Poland.

The objectives of this study were to assess the incidence of invasive pneumococcal disease (IPD) in Poland (2006-2009), where mass vaccination had not been implemented, and to determine the serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae isolates. The IPD incidence rates were highest among children under 2 years of age (3.39/100,000 in 2009) and children 2-5 years old (2.44/100,000). The most common serotypes were 14, 3, 1, 4, 19F, 23F, 6B, and 12F (61.7% of all isolates). In children aged less than 5 years, isolates of serotypes 14, 6B, and 19F were most prevalent (52.7% of the IPD cases). The PCV7, PCV10, and PCV13 covered 43.3%, 54.8%, and 68.8% of all IPD cases, and 68.7%, 76.3%, and 86.3% of cases involving children under 5 years of age. Penicillin resistance was found in 21.3% of the isolates responsible for meningitis and in 1.2% of isolates responsible for other invasive infections. Introduction of antipneumococcal conjugated vaccines into the national immunisation programme would likely lead to a significant reduction of IPD-associated morbidity among Polish children in particular, as well as in the population as a whole, especially in cases involving pneumococci with a decreased susceptibility to antibiotics.

[Invasive pneumococcal disease in the Malopolska region of Poland, in the year 2002-2008. Is introduction of mass vaccination with conjugated pneumococcal vaccine justified?]. / Inwazyjna choroba pneumokokowa w malopolsce w latach 2000-2008. Czy wprowadzenie powszechnych szczepien z zastosowaniem skoniugowanej szczepionki pneumokokowej jest zasadne?

UNLABELLED: According to the WHO pneumococcal infections are the most common cause of morbidity and mortality in children. OBJECTIVES: The aim of the study was to establish the prevalence of pneumococcal isolates belonging to serotypes covered by 7-valent conjugated pneumococcal vaccine (7vPCV) isolated from invasive pneumococcal disease (IPD) in the Malopolska region of Poland in the years 2000-2008. MATERIAL AND METHODS: Retrospective clinical and microbiological analysis was performed on invasive, laboratory confirmed pneumococcal cases in the Malopolska region, between 2000-2008. RESULTS: During the study period there were 28 cases of IPD in children under 15 years of age in the Malopolska region. Most of the cases were diagnosed as meningitis, there were also cases of bacteraemic pneumonia and sepsis. The most common serotypes found during the study were: 14, 19F, 6B and 23F. Pneumococcal 7vPCV vaccine coverage among all cases was 78.0% and among children under 5 it was 94.0%. Nine isolates (32.1%) showed decreased susceptibility to penicillin. There were three fatal cases (CFR=10.7%) due to isolates of serotypes 19F, 23 F and 6B. In the six cases of meningitis, serious and lasting sequels were observed. All complicated and fatal cases as well as the cases caused by isolates with decreased susceptibility to penicillin were caused by serotypes covered by 7vPCV. CONCLUSIONS: The most serious cases of invasive pneumococcal infections in Malopolska region were caused by isolates of serotypes covered by 7-valent conjugated pneumococcal vaccine. The results of this study prove the advantages of wide usage of the pneumococcal vaccine in the Polish population children.