Breeding of High Daptomycin-Producing Strain by Streptomycin Resistance Superposition

Daptomycin is a cyclolipopeptide antibiotic produced by Streptomyces roseosporus. It is widely used to treat drug-resistant bacterial infections; however, daptomycin yield in wild strains is very low. To improve the daptomycin production by the strain BNCC 342432, a modified method of ribosome engineering with superposition of streptomycin resistance was adopted in this study. The highest-yield mutant strain SR-2620 was obtained by increasing streptomycin resistance of BNCC 342432, and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation, 1.79-fold higher than the parent strain and its heredity stability was stable. The morphological characteristics of the two strains were significantly different, and the 440^th base G of the rpsL gene in the mutant strain was deleted, which resulted in a frameshift mutation. Our results demonstrate that gradually increasing strain resistance to streptomycin was an effective breeding method to improve daptomycin yield in S. roseosporus. Open in a separate window     

Identification and Antibiotic Susceptibility of Eggerthella Lenta in Bloodstream Infections

The identification and antibiotic susceptibility of two clinical isolates of Eggerthella lenta from bloodstream infections were determined. This microorganism is rarely pathogenic, and the findings are presented here to promote the detection and awareness of this infection. The bacteria were obtained from one patient with pressure sores and another with a malignant gastric tumor. Smears were prepared, stained, and examined by microscopy. Single colonies were analyzed by Gram staining, MALDI-TOF MS, and the 16S rRNA gene sequencing. Antibiotic sensitivity was assessed by the agar dilution method. The bacilli were found to be Gram-positive, and the MS results showed 99.8% homology with E. lenta. It was confirmed by gene sequencing. Antibiotic susceptibility tests demonstrated that E. lenta was sensitive to piperacillin-tazobactam, ampicillin-sulbactam, imipenem, meropenem, metronidazole, clindamycin, and vancomycin. This study could increase awareness of this rare infection. Open in a separate window     

Structural and Dynamic Analysis of Leaf-Associated Fungal Community of Walnut Leaves Infected by Leaf Spot Disease Based Illumina High-Throughput Sequencing Technology

Leaf-associated microbiota is vital in plant-environment interactions and is the basis for micro-ecological regulation. However, there are no studies on the direct differences in microbial community composition between disease-susceptible and healthy walnut leaves. This study collected five samples of healthy and infected leaves (all leaves with abnormal spots were considered diseased leaves) from May to October 2018. Differences in fungal diversity (Chao1 index, Shannon index, and Simpson index) and community structure were observed by sequencing and analyzing diseased and healthy leaf microbial communities by Illumina HiSeq sequencing technology. The main fungal phyla of walnut leaf-associated were Ascomycota, Basidiomycota, and Glomeromycota. Diversity indices (Shannon and Chao1 index values) of healthy leaves differed significantly in the late stages of disease onset. The results showed that the fungal species that differed considerably between the healthy and infected groups differed, and the fungal species that differed significantly between the healthy and infected groups changed with the development of the leaf disease. Critical control time points were determined by analyzing the population dynamics of pathogenic fungi. Leaf-associated microorganisms are abundant and diverse, and fungal identification and diversity studies are helpful for developing more appropriate walnut management strategies Open in a separate window     

Susceptibility of Clostridium Sporogenes Spores to Selected Reference Substances and Disinfectants

Research on the susceptibility of the spores of anaerobic bacteria such as Clostridium sporogenes or Clostridioides difficile is vital for assessing the sporicidal activity of disinfectants. The diverse susceptibility of anaerobic bacteria spores may lead to different disinfection parameters being determined by laboratories that prepare spore suspensions to test sporicidal effectiveness. The tests were performed using the suspension method according to PN-EN 13704:2018-09. In order to assess the susceptibility of the C. sporogenes spores, the criterion established for the C. difficile ribotype 027 spores was used in accordance with PN‑EN 17126:2019-01. The susceptibility of the C. sporogenes spores to glutardialdehyde corresponded to the susceptibility ranges established for the C. difficile ribotype 027 spores. The C. sporogenes spore suspension was susceptible to low concentrations of peracetic acid (0.01%). A disinfectant containing peracetic acid as the active substance showed high sporicidal activity at a low concentration (1%), a short contact time (15 minutes), and a high organic load (3.0 g/l bovine albumin + 3.0 ml/l sheep erythrocytes), as compared to a disinfectant with glutardialdehyde, which was sporicidal at a higher concentration (2.5%), at a longer contact time(60 minutes) and lower organic conditions (3.0 g/l bovine albumin). There is a need to define the minimum susceptibility criteria for the C. sporogenes spores to the reference substances most often found in disinfectants with sporicidal activity. Excessive susceptibility of the C. sporogenes spores to reference substances may result in low-performance parameters of disinfection products with sporicidal activity and lead to ineffective disinfection in practice. Open in a separate window     

Genetic Polymorphisms of Pneumocystis Jirovecii in HIV-Positive and HIV-Negative Patients in Northern China

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China. Open in a separate window     

Prevalence and Drug Resistance Pattern of Mycobacterium Tuberculosis Isolated from Tuberculosis Patients in Basra,Iraq

Drug-resistant Mycobacterium tuberculosis (DR-MTB) is a major health threat to human beings. This study aimed to evaluate the prevalence and drug resistance profile of MTB. Data were collected from 2,296 newly diagnosed, and 246 retreated tuberculosis (TB) patients who attended the Advisory Clinic for Chest Diseases and Respiratory in Basra province from January 2016 to December 2020. Both new diagnostic and retreated TB cases showed that DR-MTB cases were significantly higher at age 15–34 years, pulmonary TB, and urban residents but with no significant difference regarding gender. The drugs resistance was significantly higher among the retreated cases compared with the new diagnostic patients (20.3% vs. 2.4%, p < 0.0001), with the percentage of the resistance to first-line drugs in primary and secondary cases including isoniazid (1% and 17.1%), rifampicin (0.78% and 15.8%), ethambutol (0.56% and 8.5%), streptomycin (1.3% and 9.75%). Notice that the most common drug resistance was against streptomycin with 1.3% in new patients and against isoniazid (17.1%) in retreated patients. The rate of total drug-resistant TB, multi-drug resistant TB, mono-drug resistant TB, and rifampicin-resistant TB among new tuberculosis cases increased in this period from 2.2 to 6.7%, 0.17 to 1.6%, 0.85 to 4%, and 0.17 to 4%, with a percentage change of 204.54, 841.17, 370.58, 22.5%, respectively. The rates of poly drug-resistant TB and ethambutol-resistant-TB dropped in this period by 15.96%, and 0.7%, with a decrease from 1.19 to 1% and from 1 to 0.3%, respectively. Similarly, the increase of drug-resistant TB among secondary cases has also occurred. In conclusion, the temporal trend showed an increase in the rate of drug resistance of M. tuberculosis since 2016, with a predominant multi-drug-resistant TB and isoniazid-resistant TB. Open in a separate window     

Oral Microbiota,a Potential Determinant for the Treatment Efficacy of Gastric Helicobacter Pylori Eradication in Humans

The oral cavity serves as another reservoir for gastric Helicobacter pylori and may contribute to the failure of gastric H. pylori eradication therapy. However, changes to the oral microbial composition after gastric H. pylori eradication therapy has not yet been identified. This study aims to dissect whether the oral microbiota is involved and which bacterium mediates the clinic failure in H. pylori eradication. In the present study, the oral microorganisms from patients who had received the gastric H. pylori eradication treatment were analyzed by a high-throughput 16S rRNA deep sequencing. We found that the β diversity and composition of oral microbiota were remarkably changed in the patients who had experienced successful gastric H. pylori eradication treatment (SE group) compared to the failure group (FE group). Significantly enriched families, including Prevotellaceae, Streptococcaceae, Caulobacteraceae, and Lactobacillaceae, were detected in the SE group. In contrast, the bacterial families, such as Weeksellaceae, Neisseriaceae, Peptostreptococcaceae, Spirochaetaceae, and Veillonellaceae, were abundantly expressed in the FE group. Five operational taxonomic units (OTUs) were positively correlated with DOB values, while two OTUs exhibited negative trends. These different enriched OTUs were extensively involved in the 20 metabolic pathways. These results suggest that a balanced environment in the oral microbiota contributes to H. pylori eradication and metabolic homeostasis in humans. Our data demonstrated that the changes in oral microbiota might contribute to the therapeutic effects of antibiotic therapy. Therefore, a different therapy on the detrimental oral microbiota will increase the therapeutic efficacy of antibiotics on H. pylori infection. Open in a separate window     

Genomic Analysis of an Excellent Wine-Making Strain Oenococcus Oeni SD-2a

Oenococcus oeni is an important microorganism in wine-making-related engineering, and it improves wine quality and stability through malolactic fermentation. Although the genomes of more than 200 O. oeni strains have been sequenced, only a few include completed genome maps. Here, the genome sequence of O. oeni SD-2a, isolated from Shandong, China, has been determined. It is a fully assembled genome sequence of this strain. The complete genome is 1,989,703 bp with a G+C content of 37.8% without a plasmid. The genome includes almost all the essential genes involved in central metabolic pathways and the stress genes reported in other O. oeni strains. Some natural competence-related genes, like comEA, comEC, comFA, comG operon, and comFC, suggest that O. oeni SD-2a may have natural transformation potential. A comparative genomics analysis revealed 730 gene clusters in O. oeni SD-2a homologous to those in four other lactic acid bacteria species (O. oeni PSU-1, O. oeni CRBO-11381, Lactiplantibacillus plantarum UNQLp11, and Pediococcus pentosaceus KCCM40703). A collinearity analysis showed poor collinearity between O. oeni SD-2a andO. oeni PSU-1, indicating great differences in their evolutionary histories. The results provide general knowledge of O. oeni SD-2a and lay the foundation for specific gene function analyses. Open in a separate window     

Analysis of Gut Microbiota in Patients with Breast Cancer and Benign Breast Lesions

Breast cancer (BC) and benign breast lesions (BBLs) are common diseases in women worldwide. The gut microbiota plays a vital role in regulating breast diseases’ formation, progression, and therapy response. Hence, we explored the structure and function of gut microflora in patients with BC and BBLs. A cohort of 66 subjects was enrolled in the study. Twenty-six subjects had BC, 20 subjects had BBLs, and 20 matched healthy controls. High throughput 16S ribosomal RNA (16S rRNA) gene sequencing technology was used to determine the microbial community structure. Compared with healthy individuals, BC patients had significantly lower alpha diversity indices (Sobs index, p = 0.019; Chao1 index, p = 0.033). Sobs and Chao1 indices were also lower in patients with BBLs than healthy individuals, without statistical significance (p = 0.279, p = 0.314, respectively). Both unweighted and weighted UniFrac analysis showed that beta diversity differed significantly among the three groups (p = 3.376e–14, p < 0.001, respectively). Compared with healthy individuals, the levels of Porphyromonas and Peptoniphilus were higher in BC patients (p = 0.004, p = 0.007, respectively), whereas Escherichia and Lactobacillus were more enriched in the benign breast lesion group (p < 0.001, p = 0.011, respectively). Our study indicates that patients with BC and BBLs may undergo significant changes in intestinal microbiota. These findings can help elucidate the role of intestinal flora in BC and BBLs patients. Open in a separate window     

Antimicrobial Resistance,Virulence Factor-Encoding Genes,and Biofilm-Forming Ability of Community-Associated Uropathogenic Escherichia Coli in Western Saudi Arabia

To explore the prevalence of multidrug-resistant community-associated uropathogenic Escherichia coli (UPEC) and their virulence factors in Western Saudi Arabia. A total of 1,000 urine samples were examined for the presence of E. coli by selective plating on MacConkey, CLED, and sheep blood agar. Antimicrobial susceptibility patterns were determined using Vitek^® 2 Compact (MIC) and the disc diffusion method with Mueller-Hinton agar. Genes encoding virulence factors (kpsMTII, traT, sat, csgA, vat, and iutA) were detected by PCR. The overall prevalence of UTI-associated E. coli was low, and a higher prevalence was detected in samples of female origin. Many of the isolates exhibited resistance to norfloxacin, and 60% of the isolates showed resistance to ampicillin. No resistance to imipenem, meropenem, or ertapenem was detected. In general, half of the isolates showed multiple resistance patterns. UPEC exhibited a weak ability to form biofilms, where no correlation was observed between multidrug resistance and biofilm-forming ability. All uropathogenic E. coli isolates carried the kpsMTII, iutA, traT, and csgA genes, whereas the low number of the isolates harbored the sat and vat genes. The diversity of virulence factors harbored by community-associated UPEC may render them more virulent and further explain the recurrence/relapse cases among community-associated UITs. To the best of our knowledge, this study constitutes the first exploration of virulence, biofilm-forming ability, and its association with multidrug resistance among UPEC isolates in Saudi Arabia. Further investigations are needed to elucidate the epidemiology of community-associated UPEC in Saudi Arabia. Open in a separate window     

Screening of mcr-1 Among Gram-Negative Bacteria from Different Clinical Samples from ICU Patients in Alexandria,Egypt: One-Year Study

Antimicrobial resistance represents a global dilemma. Our present study aimed to investigate the presence of mcr-1 among different Gram-negative bacteria including Enterobacteriaceae (except intrinsically resistant to colistin) and Pseudomonas aeruginosa. Gram-negative bacterial isolates were collected from different ICUs in several Alexandria hospitals from June 2019 to June 2020. The identification of these Gram-negative isolates was made using the VITEK-2^® system (BioMérieux, France). SYBR Green-based PCR was used to screen for the presence of mcr-1 using a positive control that we amplified and sequenced earlier in our pilot study. All isolates were screened for the presence of mcr-1 regardless of their colistin susceptibility. Isolates that harbored mcr-1 were tested for colistin susceptibility and for the presence of some beta-lactamase genes. Klebsiella pneumoniae isolates harboring mcr-1 were capsule typed using the wzi sequence analysis. Four hundred eighty isolates were included in this study. Only six isolates harbored mcr-1.1. Of these, four were resistant to colistin, while two (K. pneumoniae and P. aeruginosa) were susceptible to colistin. Five of the six isolates were resistant to carbapenems. They harbored bla_OXA-48, and three of them co-harbored bla_NDM-1. K-58 was the most often found among our K. pneumoniae harboring mcr-1.1. To our knowledge, this is the first time to report colistin susceptible P. aeruginosa and K. pneumoniae harboring the mcr-1.1 gene in Egypt. Further studies are needed to investigate the presence of the mcr genes among colistin susceptible isolates to shed more light on its significance as a potential threat. Open in a separate window     

Helicobacter Pylori and Epstein-Barr Virus Co-Infection in Polish Patients with Gastric Cancer – a Pilot Study

The infectious agents may be the etiological factor of up to 15–20% of cancers. In stomach cancer, attention is paid to Helicobacter pylori and Epstein-Barr virus, both of which cause gastritis and can lead to tumor development. In co-infection, the inflammatory process is much more intense. We assessed the seroprevalence towards H. pylori and EBV in 32 patients with diagnosed gastric cancer. H. pylori antibodies were found in 69% patients, and anti-EBV – in all of them. The study confirmed that co-infection of H. pylori and EBV seems to be important in etiopathology of gastric cancer. Open in a separate window     

Impact of Primary and Secondary Bile Acids on Clostridioides Difficile Infection

Primary bile acids (BAs), synthesized from cholesterol in the liver, after their secretion with bile into the intestinal lumen, are transformed by gut microbiota to secondary BAs. As natural detergents, BAs play a key role in the digestion and absorption of lipids and liposoluble vitamins. However, they have also been recognized as important signaling molecules involved in numerous metabolic processes. The close bidirectional interactions between BAs and gut microbiota occur since BAs influence microbiota composition, whereas microbiota determines BA metabolism. In particular, it is well established that BAs modulate Clostridioides difficile life cycle in vivo. C. difficile is a cause of common nosocomial infections that have become a growing concern. The aim of this review is to summarize the current knowledge regarding the impact of BAs on the pathogenesis, prevention, and treatment of C. difficile infection. Open in a separate window     

Assessing Social – Ecological Trade-Offs to Advance Ecosystem-Based Fisheries Management

Modern resource management faces trade-offs in the provision of various ecosystem goods and services to humanity. For fisheries management to develop into an ecosystem-based approach, the goal is not only to maximize economic profits, but to consider equally important conservation and social equity goals. We introduce such a triple-bottom line approach to the management of multi-species fisheries using the Baltic Sea as a case study. We apply a coupled ecological-economic optimization model to address the actual fisheries management challenge of trading-off the recovery of collapsed cod stocks versus the health of ecologically important forage fish populations. Management strategies based on profit maximization would rebuild the cod stock to high levels but may cause the risk of stock collapse for forage species with low market value, such as Baltic sprat (Fig. 1A). Economically efficient conservation efforts to protect sprat would be borne almost exclusively by the forage fishery as sprat fishing effort and profits would strongly be reduced. Unless compensation is paid, this would challenge equity between fishing sectors (Fig. 1B). Optimizing equity while respecting sprat biomass precautionary levels would reduce potential profits of the overall Baltic fishery, but may offer an acceptable balance between overall profits, species conservation and social equity (Fig. 1C). Our case study shows a practical example of how an ecosystem-based fisheries management will be able to offer society options to solve common conflicts between different resource uses. Adding equity considerations to the traditional trade-off between economy and ecology will greatly enhance credibility and hence compliance to management decisions, a further footstep towards healthy fish stocks and sustainable fisheries in the world ocean.Open in a separate windowFigure 1Summary of multispecies management options in the Baltic.(A) Profit maximum. (B) Economic optimum while respecting sprat B_PA. (C) Equitable optimum while respecting sprat B_PA. Central numbers indicate total profits (million €/year) as well as an equity measure (in brackets). Area of each pie slice is relative to status quo values 2008-2010 (black circle), with error bars from sensitivity analysis.     

The Incubation Period of Buruli Ulcer (Mycobacterium ulcerans Infection)

Introduction

Buruli Ulcer (BU) is caused by the environmental microbe Mycobacterium ulcerans. Despite unclear transmission, contact with a BU endemic region is the key known risk factor. In Victoria, Australia, where endemic areas have been carefully mapped, we aimed to estimate the Incubation Period (IP) of BU by interviewing patients who reported defined periods of contact with an endemic area prior to BU diagnosis.

Method

A retrospective review was undertaken of 408 notifications of BU in Victoria from 2002 to 2012. Telephone interviews using a structured questionnaire and review of notification records were performed. Patients with a single visit exposure to a defined endemic area were included and the period from exposure to disease onset determined (IP).

Results

We identified 111 of 408 notified patients (27%) who had a residential address outside a known endemic area, of whom 23 (6%) reported a single visit exposure within the previous 24 months. The median age of included patients was 30 years (range: 6 to 73) and 65% were male. 61% had visited the Bellarine Peninsula, currently the most active endemic area. The median time from symptom onset to diagnosis was 71 days (range: 34–204 days). The midpoint of the reported IP range was utilized to calculate a point estimate of the IP for each case. Subsequently, the mean IP for the cohort was calculated at 135 days (IQR: 109–160; CI 95%: 113.9–156), corresponding to 4.5 months or 19.2 weeks. The shortest IP recorded was 32 days and longest 264 days (Figure 1 & 2). IP did not vary for variables investigated.Open in a separate windowFigure 1Geographic representation of Bellarine Peninsula, considered endemic for BU as of 2012.Bellarine Peninsula – east of line from Geelong to Torquay. Mornington and Westernport – southwest of line from Hampton to Tooradin (including Phillip Island).Open in a separate windowFigure 2Geographic representation of East Gippsland, considered endemic for BU as of 2012.East Gippsland: East of Sale and south of the great divide.

Conclusions

The estimated mean IP of BU in Victoria is 135 days (IQR: 109–160 days), 4.5 months. The shortest recorded was IP 34 days and longest 264 days. A greater understanding of BU IP will aid clinical risk assessment and future research.     

Splenogonadal Fusion: An Unusual Case of an Acute Scrotum

We highlight a case on a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophilic infiltrate and thrombus consistent with chronic infarction and torsion. Splenogonadal fusion (SGF) is a rather rare entity, with approximately 184 cases reported in the literature. The most comprehensive review was that of 123 cases completed by Carragher in 1990. Since then, an additional 61 cases have been reported in the scientific literature. We have studied these 61 cases in detail and have included a summary of that information here.Key words: Splenogonadal fusion, Acute scrotumA 10-year-old boy presented with worsening left-sided scrotal pain of 12 hours’ duration. The patient reported similar previous episodes occurring intermittently over the past several months. His past medical history was significant for left hip dysplasia, requiring multiple hip surgeries. On examination, he was found to have an edematous left hemiscrotum with a left testicle that was rigid, tender, and noted to be in a transverse lie. The ultrasound revealed possible polyorchism, with two testicles on the left and one on the right (Figure 1), and left epididymitis. One of the left testicles demonstrated a loss of blood flow consistent with testicular torsion (Figure 2).Open in a separate windowFigure 1Ultrasound of the left hemiscrotum reveals two spherical structures; the one on the left is heterogeneous and hyperdense in comparison to the right.Open in a separate windowFigure 2Doppler ultrasound of left hemiscrotum. No evidence of blood flow to left spherical structure.The patient was taken to the operating room for immediate scrotal exploration. A normalappearing left testicle with a normal epididymis was noted. However, two accessory structures were noted, one of which was torsed 720°; (Figure 3). An inguinal incision was then made and a third accessory structure was noted. All three structures were connected with fibrous tissue, giving a “rosary bead” appearance. The left accessory structures were removed, a left testicular biopsy was taken, and bilateral scrotal orchipexies were performed.Open in a separate windowFigure 3Torsed accessory spleen with splenogonadal fusion.Pathology revealed a normal left testicle with a fibrovascular cord with three nodules consistent with splenic tissue. The torsed splenule demonstrated hemorrhage with neutrophillic infiltrate and thrombus consistent with chronic infarction and torsion (Figure 4).Open in a separate windowFigure 4Splenogonadal fusion, continuous type with three accessory structures.     

A new species of Stenoloba Staudinger, 1892 from China (Lepidoptera,Noctuidae, Bryophilinae)

A new species of Stenoloba from the olivacea species group, Stenoloba solaris, sp. n. (Lepidoptera, Noctuidae), is described from Yunnan, China. Illustrations of the male holotype and its genitalia are provided. A diagnostic comparison is made with Stenoloba albistriata Kononenko & Ronkay, 2000, Stenoloba olivacea (Wileman, 1914), and Stenoloba benedeki Ronkay, 2001 (Fig. 4).Open in a separate windowFigures 1–5.Stenoloba spp. adults and biotope. 1 Stenoloba solaris, sp. n., male, holotypus, Yunnan, China (GBG/ZSM) 2 Stenoloba albistriata, male, paratypus, N. Vietnam (ZFMK) 3 Stenoloba olivacea, male, Taiwan (HNHM) 4 Stenoloba benedeki, male, paratypus, N. Vietnam (HNHM) 5 Type locality of Stenoloba solaris, sp. n. China, NW Yunnan, Lijiang/Zhongdian near Tuguancum, 27°29''700"N, 99°53''700"E.     

Simple,Fast, and Sensitive Method for Quantification of Tellurite in Culture Media

A fast, simple, and reliable chemical method for tellurite quantification is described. The procedure is based on the NaBH₄-mediated reduction of TeO₃²⁻ followed by the spectrophotometric determination of elemental tellurium in solution. The method is highly reproducible, is stable at different pH values, and exhibits linearity over a broad range of tellurite concentrations.The tellurium oxyanion tellurite is toxic for most organisms, making important its accurate assessment. Several methods for quantifying tellurite have been described to date. However, most of them are rather complicated and require sophisticated equipment and in some cases the detection is not quite sensitive enough to allow the assessment of TeO₃²⁻ concentrations below 50 μg/ml (200 μM). For example, the analytical determination of tellurium (Te) oxyanions by atomic absorption spectrometry (AAS) is hampered by poor sensitivity. Where flame or electrothermal AAS routinely yields detection limits of less than 10 ppb for iron (16), normal flame AAS tellurium detection limits are 100 to 1,000 times higher and require pretreatment to achieve the +IV oxidation state before analysis (11).On the other hand, hydride generation AAS (HGAAS) is used to achieve ppb-level detection limits for Se and Te as well as arsenic and antimony among others. For Te the volatile hydride gas H₂Te is generated by first converting the metalloid to the +IV oxidation state and then by chemical reduction to the gaseous hydride using—almost universally—sodium borohydride (NaBH₄). In automated HGAAS systems, an inert purge gas sweeps the volatile hydride formed in a glass reaction vessel into a quartz cell heated by the AAS flame where gaseous hydride decomposition and atomization occur. Though tellurite reduction, precipitation, and detection methods have been reported (3, 17), they are temporally relatively unstable and pH dependent.Since tellurium is toxic and environmentally important (7, 8), determining low concentrations in bacterial cultures is very desirable and a simple analysis without pretreatment steps that could quickly establish total metalloid oxyanion content in a liquid sample would be a plus. Here we report a new method for the determination of tellurite in bacterial culture media. This procedure is based on the NaBH₄ reduction of tellurite to the elemental form, which is analyzed spectrophotometrically at 500 nm or 320 nm (see below), by which the light scattered by the particles of elemental metalloid in solution is measured. While the detection limits do not compare to those of HGAAS (14) or capillary electrophoresis (13), they do approach those of old flame AAS but involve a much simpler and quicker procedure requiring only one reagent and a spectrophotometer to determine total content of solutions of +IV oxyanions in solution. Linear calibration range, method development time and probe stability, effect of sample pH, common interferences, and detection limits were investigated.Calibration curves to determine K₂TeO₃ concentrations in routinely used microbiological culture media such as Luria-Bertani (LB) or M9 minimal medium amended with 0.2% glucose (15) were constructed. A set of solutions containing increasing concentrations of K₂TeO₃ (Sigma) were prepared in LB or M9 culture medium, and the tellurium oxyanion was quantitatively reduced using freshly prepared 3.5 mM NaBH₄ (final concentration).The reaction was carried out at 60°C for 10 min (bubbling was overcome by vortexing), and after 5 min at room temperature, the optical density at 500 nm (OD₅₀₀) was determined spectrophotometrically as described previously (4, 5, 9, 12). Blanks contained no borohydride. Figure ​Figure11 shows that in both media good curve linearity was obtained, with r² values of 0.9740 and 0.9963 for LB and M9, respectively. Tellurite concentrations lower than 1 μg/ml or higher than 200 μg/ml were also tested, but OD₅₀₀ values were close to the spectrophotometer error limit at low concentrations or nonlinear above 200 μg/ml (not shown). Thus, the NaBH₄ method allows determination of a wide range of tellurite concentrations in a fast and simple way. Tellurite concentrations lower than 50 μg/ml in both rich and minimal media can be easily determined; the experimental error was about 10%, similar to that reported for the diethyl dithiocarbamate (DDTC) tellurite method (17).Open in a separate windowFIG. 1.Calibration curves to determine K₂TeO₃ concentrations in LB (A) (R² = 0.9963) or M9 minimal (B) (R² = 0.9740) medium. Optical density at 500 nm was determined after reducing the oxyanion with sodium borohydride. Error bars denote 1 standard deviation of three replicates.To analyze the resulting solutions after tellurite reduction by NaBH₄, absorption/scattering spectra were determined. Figure ​Figure22 shows that spectra from LB and those from M9 after tellurite reduction are quite different, which may be a consequence of the different chemical compositions of these culture media. In both cases, absorption spectra showed linearity between optical density at 500 nm and tellurite concentration in the sample. However, high tellurite concentrations (∼100 μg/ml) caused a loss of linearity in LB medium.Open in a separate windowFIG. 2.Absorption spectra after reducing samples of LB (A) or M9 (B) culture medium containing increasing tellurite concentrations with 3.5 mM NaBH₄. Tellurite concentrations used were 20, 40, 60, 80, and 100 (LB) and 2, 4, 6, 8, and 10 (M9) μg/ml. (Inset) Calibration curve in M9 medium using the absorbance maxima at 320 nm.Figure ​Figure2B2B shows that in M9 medium there is a zone around 320 nm exhibiting higher optical density than that at 500 nm, which represents an advantage in the determination of tellurite in chemically defined culture media. This is reflected in a wider range of measurable concentrations at 320 nm (Fig. ​(Fig.2B,2B, inset), as well as in a higher sensitivity of the method as determined by the slope of the calibration curve. The product of tellurite reduction by NaBH₄ showed good stability at both wavelengths in rich and minimal culture media (not shown).Since in M9 medium the method allows the determination of minor tellurite concentrations (1 to 20 μg/ml), it would be of great help in assessing tellurite uptake in tellurite-sensitive microorganisms whose MICs range from 1 to 10 μg/ml. Sulfur-containing salts, commonly present in culture media as sulfites and sulfates, did not interfere with our NaBH₄ method for tellurite assessment at concentrations up to 0.5 M (not shown).As shown in Fig. ​Fig.3,3, tellurite assessment was not affected by the pH of the culture medium. In fact, linearity was observed in a wide pH range with minor slope changes in LB. Similar results were obtained with M9 medium, although tellurite assessment was not possible at pH values higher than 7.0 because of the formation of a precipitate. This may be due to an interaction of the phosphate salts present in the medium and some charged (2⁺) chemical species forming at alkaline pH values, as has been reported earlier (17).Open in a separate windowFIG. 3.Effect of pH in determining tellurite concentrations in LB (A) and M9 minimal (B) media.To date, the most commonly used procedure for determining tellurite in culture media is that involving the spectrophotometric determination (340 nm) of the complex that forms between tellurite and diethyl dithiocarbamate (17). This procedure has been used to assess tellurite uptake by the phototrophic bacterium Rhodobacter capsulatus, which is naturally resistant to K₂TeO₃ (MIC, ∼1.4 mM) (2, 3). However, K₂TeO₃ uptake studies in highly sensitive cells such as Escherichia coli (MIC, ∼4 μM) are difficult to carry out because of the low concentrations of toxicant present in the culture medium, far below the detection limit of the DDTC procedure (17).In this context and for testing the applicability of our method in vivo, we used the tellurite-sensitive bacterium E. coli BW25113 (10) and the tellurite-resistant Aeromonas caviae ST (5, 6). An overnight culture of E. coli BW25113 in M9 minimal medium was diluted 100-fold with fresh M9 supplemented with 0.2% glucose and grown at 37°C with shaking. When the OD₆₀₀ was 0.1, the culture was amended with 20 μg/ml K₂TeO₃ (arrow, Fig. ​Fig.4A).4A). Then aliquots were taken at the indicated times and cells were centrifuged at 8,500 × g for 3 min; supernatants were used to assess extracellular tellurite by our NaBH₄ method. While added tellurite did not affect bacterial growth (Fig. ​(Fig.4A),4A), the remaining tellurite in the supernatant dropped approximately to one-third after 3 h (Fig. ​(Fig.4B).4B). Tellurite determinations were validated using, in parallel, the DDTC method (not shown).Open in a separate windowFIG. 4.Tellurite uptake by Escherichia coli. Time zero in panel B represents the moment of tellurite addition.Regarding the tellurite-resistant bacterium A. caviae ST, a 1:100 dilution of an overnight culture was inoculated into fresh LB medium and the OD₆₀₀ was recorded at the indicated times. When the OD₆₀₀ was ∼0.4, the culture was amended with tellurite (400 μg/ml final concentration) (Fig. ​(Fig.5A,5A, arrow) and the remaining tellurite in the supernatants was assessed as described above. Figure ​Figure5B5B shows that in 4 h ∼27% of the toxic oxyanion was removed from the culture medium.Open in a separate windowFIG. 5.Tellurite uptake by Aeromonas caviae ST. See the text for details.In summary and in comparison to the DDTC procedure, the NaBH₄ method described here allows more sensitive determination of the initial tellurite concentrations as well as the continuous uptake of the toxicant by tellurite-sensitive and tellurite-resistant microorganisms. This method should be of great help in future studies aimed at unveiling the tellurite reductase activity exhibited by some metabolic enzymes such as nitrate reductase (1), catalase (4), and the pyruvate dehydrogenase complex (5, 6). These studies are currently being carried out in our laboratory.