In studies from other African settings, hepatotoxicity from TB therapy has been reported to be low [27, 28]. In Tanzania, the prevalence of hepatotoxicity was only 0.9% at 2 months of TB therapy [27]. Similarly, in Malawi, among HIV-infected ART-naïve patients during TB treatment, only five (1.3%) developed grade 2 hepatotoxicity (defined as ALT = 126–250 IU/L), three (0.9%) developed grade 3 hepatotoxicity (defined as ALT = 251–500 IU/L) and there was no grade 4 hepatotoxicity (defined as ALT > 500 IU/L) [28]. Breen et al. found serious adverse events of TB therapy in 40% of HIV-infected patients, U0126 71% of whom were on concomitant ART, as opposed to only 26% of HIV uninfected patients (P = 0.008). However, the

rate of hepatotoxicity was comparable between the two groups [29]. Therefore, it is likely that the risk of hepatotoxicity with anti-TB therapy observed among HIV-infected individuals is a result of interaction or confounding with other risk factors such as hepatitis C, hepatitis B or ART treatment and not HIV infection per se, as has been previously suggested [30]. Our study had several limitations. First, we did not collect data on illicit drugs or alcohol consumption, which are important risks for elevated

ALT. Secondly, we were unable to include 37% of patients otherwise eligible for our programme, either because they were non-ART-naïve at enrolment (10%) or because of missing baseline ALT measurements (27%). Patients included in this analysis were sicker with more advanced AP24534 mouse HIV infection. Our study and others published in the literature have found that the risk of elevated ALT is higher in patients with more advanced HIV disease. Thus, the prevalence of elevated ALT may be somewhat overestimated in this report. However, there was also a small significant

difference in the distribution by district, but is not clear how district would affect the prevalence of estimates. Regardless of the district, all clinics included in this analysis are supported by the same programme, MDH-PEPFAR, which offers similar care to patients. It is important to emphasize that these small differences in baseline characteristics between the patients included in this analysis and those excluded are not expected to interfere with the internal validity of this analysis, particularly concerning the risk factors identified in Table 2. Thirdly, because this study was cross-sectional, the temporal sequence of exposure and outcome cannot be ascertained. A longitudinal design would allow for a more precise determination of predictors of elevated ALT. Use of a laboratory surrogate marker (i.e. elevated ALT level) as a sign for hepatopathy is less sensitive than other noninvasive and invasive measures of detecting liver disease, such as Fibroscan® (ECHOSENS; Paris, France) and liver biopsy. However, these investigations are neither available nor feasible in the study setting.

uAPRs were available in 144 patients: 46 patients (32%) had TP and 21 (15%) GP; the remainder had uPCR < 30 mg/mmol. The TP Navitoclax ic50 group had a higher fractional excretion of phosphate compared with the GP group (mean 27% vs. 16%, respectively; P < 0.01). Patients with TP were more likely to be on tenofovir and/or a boosted

protease inhibitor compared with those with GP. In 18 patients with heavy proteinuria (uPCR > 100 mg/mmol), a renal assessment was made; eight had a kidney biopsy. In all cases, the uAPR results correlated with the nephrological diagnosis. In HIV-infected patients, measuring uAPR may help to identify patients in whom a renal biopsy is indicated, and those in whom tubular dysfunction might be an important cause of proteinuria and which may be related to antiretroviral toxicity. We suggest that this would be useful as a routine screening procedure in patients with proteinuria. A spectrum of renal disease occurs in HIV-infected patients [1]. Chronic kidney disease (CKD) can be caused by the virus itself, sometimes manifesting as HIV-associated nephropathy (HIVAN) or HIV-associated immune complex kidney disease (HIVICK) [2-4]. Alternatively and increasingly, it Fostamatinib is attributable to other unrelated pathologies,

for example, hypertension, diabetes, opportunistic infections or other viral coinfections [5, 6], and it is becoming more important to identify this group. Renal disease can also be caused by combination antiretroviral therapy (cART) [1]. As survival in HIV-infected patients improves, interest in cART-related renal toxicity continues to grow. Tenofovir (TDF) is a nucleotide reverse

transcriptase inhibitor that is an effective antiretroviral drug widely used as first-line treatment [7]. Although some data suggest that it is not reliably associated with increased renal toxicity [8-11], there are increasing numbers of reports and studies of renal tubular dysfunction, with rare reports of Fanconi syndrome [12-15]. Data from other studies confirm that TDF co-prescribed with a boosted protease inhibitor (PI) is associated with the highest risk of such toxicity [16-18]. Screening for proteinuria in HIV-infected patients is therefore important, as it is often an early indicator of underlying kidney dysfunction. Orotidine 5′-phosphate decarboxylase There are different methods for routinely assessing proteinuria. How, and when, to screen for proteinuria continues to be debated. Urine dipstick analysis is frequently performed, but in the context of urine protein, it mainly detects albumin and may fail to identify those patients in whom protein in the urine is predominantly caused by other proteins. It is generally accepted that measurement of the urine protein/creatinine ratio (uPCR) and the urine albumin/creatinine ratio (uACR) is a relatively cheap (approximately £0.20 and £0.50, respectively) and effective way to screen for renal disease [19]. The specific test used often depends upon the laboratory practice.

In addition, Dr Grietje Holtrop (Biomathematics and Statistics Scotland) provided valuable input in the statistical analysis of data. The work described in this manuscript was supported by a grant received from the Food Standards Agency (FSA; G03031). The Rowett Institute of Nutrition and Health receives support from the Scottish Government (Rural and Environment Science and Hydroxychloroquine Analytical Services; RESAS). “
“Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published

reports describing the application of asRNA gene silencing for comprehensive analyses learn more of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250 000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E. coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential

genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E. coli using regulated asRNA expression. During the past few decades, bacterial pathogens have become

increasingly resistant to antibiotics, limiting treatment options for infections caused by drug-resistant bacterial pathogens (Boucher et al., 2009). As we face growing antibiotic resistance, the development of novel antibiotics continues to stagnate. Therefore, there is an urgent need for the discovery of new antibacterial agents to target drug-resistant bacteria, especially C1GALT1 Gram-negative pathogens (Boucher et al., 2009). Regulated antisense RNA (asRNA) expression has been used effectively to study gene functions in different bacterial systems, including Streptococcus mutans (Wang & Kuramitsu, 2005), Staphylococcus aureus (Ji et al., 2001; Forsyth et al., 2002), and Escherichia coli (Nakashima & Tamura, 2009). By blocking the expression of its target gene, an asRNA increases the sensitivity of bacteria only to specific inhibitors for a protein encoded by that target gene (Forsyth et al., 2002; Young et al., 2006).

, 2011). 3ADON chemotype synthesizes

DON and 3ADON, 15ADON chemotype produces DON and 15ADON, while NIV chemotype produces NIV and 4ANIV (4-acetylnivalenol; selleckchem Wang et al., 2011). However, it has been documented that some isolates from one defined chemotype are able to produce mycotoxins from other chemotypes in considerable amounts (Ward et al., 2002; Mugrabi de Kuppler et al., 2011). In F. graminearum, the enzymes catalyzing the biochemical reactions which result in formation of trichothecenes are encoded by tri genes (Foroud & Eudes, 2009). Polymorphism of tri sequences contributes to the trichothecene chemotypes. NIV synthesis is determined by the expression of both tri7 and tri13 genes, while in DON chemotypes, tri13 and tri7 are nonfunctional as a result of multiple insertions and AZD6244 purchase deletions (Lee et al., 2002). The sequence differences resulting in differential activity of tri8 are a key determinant of the 3ADON and 15ADON chemotypes in F. graminearum (Alexander et al., 2011). Besides its genetic background, mycotoxin production has received considerable attention in analyses of external factors affecting trichothecene production within Fusarium. It has been demonstrated that regulation of mycotoxin biosynthesis occurs primarily at a transcriptional level (Proctor et al., 1999; Marín et al., 2010). Estimating

relative transcript abundances by RT-qPCR allows for precise identification of factors regulating the biosynthesis of mycotoxins in Fusarium (Merhej et al., 2011). The impact of abiotic factors such as temperature (Schmidt-Heydt et al., 2008; Marín et al., 2010), osmotic potential (Marín et al., 2010), and pH (Merhej et al., 2010) on tri transcript levels and trichothecene accumulation in media has been examined. Moreover, several reports have indicated that different substrates (Jiao et al., 2008; Gardiner et al., 2009) and signaling molecules (Ponts et al., 2007) regulate mycotoxin production in Fusarium. Limited studies Anacetrapib have identified the

impact of anthropogenic factors such as fungicides on trichothecene biosynthesis within Fusarium, especially at a transcriptional level (Covarelli et al., 2004; Ochiai et al., 2007). Among the fungicides used, the application of azoles during wheat anthesis is a primary method for management of FHB (Paul et al., 2010). These compounds block the ergosterol biosynthesis pathway by inhibiting the sterol 14- α -demethylase encoded by the CYP51 gene (Liu et al., 2010). Azoles have been shown to be effective in reducing FHB symptoms and DON content in wheat, although the effectiveness between azole compounds varies (Paul et al., 2010). On the other hand, unsatisfactory effects of this group of fungicides against Fusarium spp. have also been documented (Mesterházy et al., 2011).

This is the first report identifying carotenoids produced by the fungus and characterizing carotenoid biosynthesis genes in the fungus. GzCarRA exhibits high sequence similarity to CarRA of F. fujikuroi (Linnemannstöns et al., 2002) and Al-2 of N. crassa (Arrach et al., 2002). These genes encode a bifunctional enzyme with both phytoene synthase and carotene cyclase activity. Our study showed that ΔgzcarRA does not produce phytoene, suggesting that GzCarRA is required for phytoene synthesis, and the high sequence similarity between GzCarRA and CarRA suggests

that GzCarRA also has cartotene cyclase activity. GzCarB is highly similar to the CarB gene of F. fujikuroi (Linnemannstöns et al., 2002), and Al-1 of N. crassa (Schmidhauser et al., 1990). Al-1 synthesizes 3,4-didehydrolycopene by introducing double bonds to the phytoene substrate via phytofluene, ɛ-carotene, neurosporene, and lycopene. The major products Apoptosis inhibitor of this enzyme are 3,4-didehydrolycopene and lycopene. Hydroxychloroquine concentration γ-Carotene is not the substrate of Al-1, suggesting that torulene is synthesized from 3,4-didehydrolycopene (Hausmann & Sandmann, 2000). In our study, ΔgzcarB accumulated phytoene, indicating that GzCarB also plays a role in the dehydrogenation of

phytoene. Therefore, we deduced that GzCarB is a phytoene dehydrogenase that catalyzes the formation of 3,4-didehydrolycopene and lycopene (Fig. 4). GzCarX and GzCarO show high similarity to carotenoid cleavage oxygenase CarX (Thewes et al., 2005) and opsin-like protein CarO (Prado et al., 2004), respectively, from F. fujikuroi. CarX expressed in Escherichia coli synthesizes retinal from β-carotene, γ-carotene, β-apo-8′-carotenal, and torulene, indicating that the function of CarX is in retinal biosynthesis (Prado-Cabrero et al., 2007b). Opsins are a class of retinal-binding proteins with seven transmembrane helical domains. In this study,

G. zeae did not produce retinal and neither ΔgzcarX nor ΔgzcarO affected neurosporaxanthin and torulene production, suggesting that both genes are not functional in the fungus. GzCarT is highly similar to CarT in F. fujikuroi. CarT functions as a torulene oxygenase, given its catalysis of the conversion of torulene into β-apo-4′-carotenal in vitro and the accumulation of torulene by the CarT null mutant of F. fujikuroi (Prado-Cabrero et Fludarabine cell line al., 2007a). As expected, ΔgzcarT also accumulated torulene and produced no neurosporaxanthin, suggesting that GzCarT is a torulene oxygenase. Based on our results, we propose the following neurosporaxanthin biosynthetic pathway in G. zeae (Fig. 4). Torulene is first synthesized by GzCarRA and GzCarB. The colorless carotenoid phytoene is synthesized from two molecules of GGPP by GzCarRA. GzCarRA is a bifunctional enzyme that contains two domains: one catalyzing phytoene synthesis and the other catalyzing the formation of β-ionone rings.

α1,6-linked) on the ability of S. mutans to form biofilms (Banas & Vickerman, 2003; Banas et al., 2007; Lynch et al., 2007; Klein et al., 2009; Koo et al., 2010). Recent studies by us and some other labs have generated evidence that alteration LDK378 in vitro of the glucans’ structure and/or the ratio of glucans to glucan-binding proteins could have a significant effect on S. mutans adherence and accumulation on a surface (Hazlett et al., 1998; Hazlett et al., 1999; Wen et al., 2005), although the basis for this phenomenon remains unclear. Similarly, aberrant expression of GtfC in TW239 would likely cause alterations in glucan structures (more α1,6-linked, water-soluble

glucans than the wild-type) and probably the ratio of

glucans to glucan-binding proteins, possibly contributing to the observed decreases in biofilm formation by the deficient mutant. In summary, this study clearly showed that the transcriptional repressor Rex plays a significant role in the regulation of central metabolism and energy generation, NAD+ regeneration, oxidative homeostasis and biofilm formation by S. mutans. Current efforts are directed toward further investigation of the underlying mechanism of Rex-mediated regulation in oxidative stress response and biofilm formation. This work is supported in part by NIDCR grants DE13239 and DE19106 to R.A.B. and DE19452 to Z.T.W. and by South Louisiana Institute of Infectious Disease Research. Table S1. Up- and downregulated genes identified by DNA microarray analysis. Please note: Wiley-Blackwell Kinase Inhibitor Library research buy is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these Florfenicol T3SSs are delivered into host cells, leading

to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus.

Known in North America since the 1920s, presumably having been accidentally introduced from its assumed East Asian centre of origin, until LDE225 solubility dmso recently, this pathogen has not been identified causing disease in Europe except for a few isolated outbreaks. However, since 2010, there have been several reports of infection of C. lawsoniana by P. lateralis in the United Kingdom, including Northern Ireland. We sequenced

the genomes of four isolates of P. lateralis from two sites in Northern Ireland in 2011. Comparison with the closely related tree and shrub pathogen P. ramorum (cause of ramorum disease of larch and other species in the UK) shows that P. lateralis shares 91.47% nucleotide sequence identity over the core conserved compartments of the genome. The genomes of the four Northern Ireland isolates are almost identical, but we identified several single-nucleotide polymorphisms (SNPs) that distinguish

between isolates, thereby presenting potential molecular markers of use for tracking routes of spread and in epidemiological studies. Our data reveal very low rates of heterozygosity (compared with P. ramorum), consistent with inbreeding within this P. lateralis population. “
“Pseudomonas aeruginosa biofilm formation was increased by addition of sucrose to Luria–Bertani medium, whereas addition of NaCl to a final similar osmolarity and use of maltose instead of sucrose, were ineffective. In a previous study, we showed that the extracytoplasmic sigma factor SigX is activated in NVP-BKM120 supplier the presence of sucrose. The sucrose-mediated pellicle increase was abolished in a sigX mutant strain. Sucrose addition led to an increase in pel expression and cyclic-diguanylate (c-di-GMP) pool level production.

Interestingly, these two phenotypes were strongly decreased in a sigX mutant. Since pel is not known as a SigX-target, we suspect SigX to be involved in the c-di-GMP production. We found that expression of the diguanylate cyclase PA4843 gene was increased in the presence of sucrose at least partly through SigX activity. Our study shows that sucrose itself rather than osmolarity favours the biofilm mode of P. aeruginosa through the activation of SigX. “
“Extensive diglyceride denitrification resulted in a dramatic increase in pH (from 6.8 to 9.5) in nitrate-impacted, acetate-amended sediment microcosms containing sediment representative of the Sellafield nuclear facility, UK. Denitrification was followed by Fe(III) reduction, indicating the presence of alkali-tolerant, metal-reducing bacteria. A close relative (99% 16S rRNA gene sequence homology) to Serratia liquefaciens dominated progressive enrichment cultures containing Fe(III)-citrate as the sole electron acceptor at pH 9 and was isolated aerobically using solid media.

Smoking is the most prevalent, modifiable, independent risk factor for CVD in HIV-infected patients [36]. As well as reducing the risk of CVD, these changes also help reduce the risk of progression to diabetes [37]. In high-risk patients, i.e. AZD8055 patients for whom the 10-year risk of CVD is ≥20%, ART modification should be considered, together with specific interventions focused on the principal risk factors for CVD, namely blood pressure, coagulation, and glucose and lipid levels. Similarly, the presence of established CVD or diabetes should also prompt the initiation of lipid-modifying therapy [5]. Impaired glucose tolerance [fasting plasma glucose

<7.0 mmol/L (126 mg/dL)] and impaired fasting glucose [fasting

plasma glucose 6.1–6.9 mmol/L (110–125 mg/dL)] increase the risk of developing diabetes four- to sixfold and increase cardiovascular morbidity and mortality [32]. Patients with glucose abnormalities should be counselled regarding lifestyle changes (Table 2) and those with diabetes [fasting plasma glucose ≥7.0 mmol/L (126 mg/dL) or oral glucose tolerance (2-h value) of ≥11.1 mmol/L (200 mg/dL)] should receive an oral anti-diabetic agent. Metformin is recommended as first-line oral anti-diabetic therapy with the addition of pioglitazone as the preferred FK228 choice for combination therapy if glycated haemoglobin (HbA1c) remains >6.5–7.0% [5]. Blood lipids and blood pressure should be carefully monitored and, where necessary, individuals should be referred

for screening for nephropathy, polyneuropathy and retinopathy. Failure to achieve a target HbA1c of <6.5–7.0% should prompt referral to a diabetes specialist for initiation of insulin therapy [5]. Early screening is not just relevant to metabolic diseases. HIV-infected patients at risk of kidney disease also benefit from early identification and referral [38]. Guidelines from the HIV Medicine Association of the Infectious Diseases Society of America (IDSA) [38] recommend that assessment for existing kidney disease, with a screening urine analysis for proteinuria, a blood test for serum creatinine and a calculated estimate of renal function, should be carried out at the time of HIV Farnesyltransferase diagnosis. The recently published EACS guidelines (see ref. 5, p. 36) highlight the potential use of urinary albumin creatinine (UA/C) or urinary albumin protein (UA/P) ratios for screening all patients and assessment of estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease (MDRD) tool developed by the Copenhagen HIV Group (see http://www.cphiv.dk/tools). Both IDSA and EACS guidelines recommend that high-risk HIV-infected patients with proteinuria and/or GFR <60 mL/min are referred to a nephrologist [5,38].

Bonarek, F. Bonnal, F. Bonnet, N. Bernard, O. Caubet, L. Caunègre, C. Cazanave, J. Ceccaldi, FA Dauchy, C. De La Taille, S. De Witte, M. Dupon, P. Duffau, H. Dutronc, S. Farbos, MC Gemain, C. Greib, D. Lacoste, S. Lafarie-Castet, P. Loste, D. Malvy, P. Mercié,

P. Morlat, D. Neau, A. Ochoa, JL. Pellegrin, JM. Ragnaud, S. Tchamgoué, JF. Viallard. Immunology: I. Pellegrin, P. Blanco, JF. Moreau. Virology: H. Fleury, ME. Lafon, B. Masquelier. Pharmacology: D. Breilh. Pharmacovigilance: G. Miremont-Salamé. Data Fer-1 collection: MJ. Blaizeau, M. Decoin, S. Delveaux, S. Gillet, C. Hannapier, S. Labarrère, V. Lavignolle-Aurillac, B. Uwamaliya-Nziyumvira. Data management: S. Geffard, G. Palmer, D. Touchard. “
“The aim of the paper was to describe the association of religion with HIV outcomes in newly diagnosed Africans living in London. A survey of newly diagnosed HIV-positive Africans attending 15 HIV treatment centres across London was carried out between April 2004 and February 2006. Confidential self-completed questionnaires were used, linked to clinical records. Bivariate analyses were conducted to ascertain

whether religious beliefs were associated with late diagnosis, antiretroviral therapy, and immunological and virological outcome 6 months post diagnosis. A total of 246 Black Africans were eligible MK-2206 cell line and included in the analysis: 62.6% were women, and the median age was 34 years. The median CD4 count at diagnosis was 194 cells/μL (range 0–1334 cells/μL) and 75.6% presented late, as defined as a CD4 count < 350 cells/μL. Most participants were religious: non-Roman Dimethyl sulfoxide Catholic Christians (55.7%), Roman Catholics (35.2%) and Muslims (6.1%). Only 1.2% stated that they did not have a religion. Participants who attended religious services at least monthly were more likely to believe that ‘faith alone can cure HIV‘ than those who attended less frequently (37.7% vs. 15.0%; P = 0.002). A small proportion (5.2%) believed that taking antiretroviral therapy implied a lack of faith in God. Bivariate analysis found no relationship between religiousness (as measured using frequency of attendance at religious

services and religious attitudes or beliefs) and late diagnosis, changes in CD4 count/viral load 6 months post diagnosis, or initiation of antiretroviral therapy. Strong religious beliefs about faith and healing are unlikely to act as a barrier to accessing HIV testing or antiretroviral treatment for Black Africans living in London. Although men who have sex with men remain the largest group affected by HIV in the UK, heterosexual Black Africans bear a disproportionate burden of the HIV epidemic in the UK [1]. In 2009, Black Africans accounted for just over a third (33.8%) of all new HIV diagnoses and 63% of heterosexuals diagnosed with HIV infection in the UK [1]. Approximately one third (32.2%) of HIV-positive Black Africans are living with undiagnosed HIV infection.

In 26 cases (0.7%), the sequences were taxonomically misclassified, Pifithrin �� representing SSU rRNA genes from other taxonomic domains. In 28 cases (0.7%), the sequences were chimeric, some of which were sequences with serious anomalies (Fig. S1c). Eight sequences (0.2%) were of poor quality (i.e. many ambiguous base calls or long homopolymers) and two queries (0.1%) exclusively contained sequences identified as cloning vectors. The remaining 101 cases (2.6%) did not show any anomaly within the scope of this investigation

and likely represented highly divergent sequences. The following reasons accounted for at least one HMM detection in both orientations, leading to the 185 sequences being flagged as uncertain. In 61 cases (33%), the sequences were reverse complementary chimeras, this website with the reverse complement segment matching one or more HMMs. In 29 cases (16%), the sequences showed only partial, poor or no match to any entry in GenBank as assessed through blast. The remaining 95 sequences (51%) did not show any anomaly within the scope of this investigation and likely represent rare false

detection by individual HMMs. In all these 95 cases, only single HMMs were detected in the opposite orientation, while the remaining HMMs were detected in the other orientation, leaving no doubt about the true orientation of the sequence (i.e. 90 forward and five reverse complementary). In conclusion, the queries showing multiple HMM detections in both orientations were all identified as having some sort of anomaly, whereas

all other queries flagged as uncertain represented rare single false-positive detections, which did not impair determination of the true orientation of the sequence. Among the 1 167 613 sequences with unambiguous orientation assignments, 3117 sequences had unusually low HMM counts of three or fewer. After looking in more detail at all these cases, we identified the following reasons for these observations. In 1882 cases (60%), the sequences contained only partial 16S information and partial up- or downstream regions, i.e. 101 upstream and 1781 downstream cases. A total of 714 sequences (23%) showed only partial, poor or no match to any entry triclocarban in GenBank, whereas 277 sequences (9%) were of poor quality. In 110 cases (4%), the sequences had been associated with wrong taxa and represented different domains, and three cases (0.1%) were chimeric sequences that contained two concatenated identical segments. The remaining 131 cases (4%) did not show any anomaly within the scope of this investigation and are likely sequences with long hypervariable regions and/or sequences that contain divergent segments that are not detected by some individual HMMs.