Assessment of microbial contamination in laser materials processing laboratories used for prototyping of biomedical devices.

Microbial contamination of medical devices during pilot production can be a significant barrier as the laboratory environment is a source of contamination. There is limited information on microbial contaminants in laser laboratories and environments involved in the pilot production of medical devices. This study aimed to determine the bioburden and microbial contaminants present in three laser laboratories - an ISO class 7 clean room, a pilot line facility and a standard laser laboratory. Microbiological air sampling was by passive air sampling using settle plates and the identity of isolates was confirmed by DNA sequencing. Particulate matter was analysed using a portable optical particle counter. Twenty bacterial and 16 fungal genera were isolated, with the genera Staphylococcus and Micrococcus being predominant. Most isolates are associated with skin, mouth, or upper respiratory tract. There was no significant correlation between microbial count and PM2.5 concentration in the three laboratories. There were low levels but diverse microbial population in the laser-processing environments. Pathogenic bacteria such as Acinetobacter baumannii and Candida parapsilosis were isolated in those environments. These results provide data that will be useful for developing a contamination control plan for controlling microbial contamination and facilitating advanced manufacturing of laser-based pilot production of medical devices.

Antimicrobial resistance in urinary pathogens and culture-independent detection of trimethoprim resistance in urine from patients with urinary tract infection.

BACKGROUND: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture. METHODS: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94). RESULTS: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC90 > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%). CONCLUSIONS: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing.

Genetic diversity and whole genome sequence analysis data of multidrug resistant atypical enteropathogenic Escherichia coli O177 strains: An assessment of food safety and public health implications.

Atypical enteropathogenic E. coli (aEPEC) strains are emerging pathogens responsible for fatal diarrhoea in humans worldwide. The purpose of this study was to investigate genetic diversity, virulence and antimicrobial resistance profiles of aEPEC O177 strains isolated from faeces of cattle reared in intensive and extensive production systems in South Africa. A total of 96 multidrug resistant (MDR) aEPEC O177 isolates were typed using enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphism DNA (RAPD) typing. The resistome, virulome and mobilome of two aEPEC O177 isolates were investigated using WGS analysis. The ERIC typing was efficient and reproducible with a discriminatory index of 0.95. RAPD typing had poor reproducibility with satisfactory discriminatory power of 0.859. The dendrograms constructed based on ERIC and RAPD banding patterns produced 9 and 8 clusters, respectively, which indicate genetic variation among E. coli O177 isolates. WGS analysis revealed that CF-154-A and CF-335-B) isolates belonged to the O177 serotype with H7 and H21, respectively. Both isolates harboured several virulome genes such as intimin (eaeA), haemolysin (hlyA and hlyE), translocated iron receptor (tir), Type III secretion system (eprH, gspL and prgH), bssR and bssS. However, genes encoding shiga toxins were not found in either isolate. Antibiotic resistance genes such as ampC, tet, ermB, sul2, strB AcrD, aph(6)-Ic, aph(6)-Ib, aph(3″)-I, ant (3″)-1a AcrA and acrE were found in the E. coli O177 strains. Furthermore, genome annotation results indicated that both isolates carried plasmids, insertion sequences, prophages and cluster of regularly interspaced short palindromic repeats (CRISPR) type I. Based on in silico multi locus typing (MLST) analysis, the two isolates were assigned to different sequence types (CF-154-A, ST-1308 and CF-335-B, ST-58). Whole genome multi locus typing tree showed that our isolates clustered with E. coli O177:H21 (reference), suggesting the close genomic relatedness among the strains. Overall, these findings showed that cattle carry genetically diverse E. coli O177 strains, which harbour a repertoire of virulome, resistome and mobilome genes. This highlights a need for multidrug resistant E. coli O177 strain surveillance in cattle.

Mycotoxins in poultry feed and feed ingredients in Nigeria.

Mycotoxins are toxic secondary fungal metabolites that can negatively affect animal productivity when ingested through feed. In order to assess mycotoxin contamination of poultry feed and feed ingredients vis-a-vis source tracking of feed contamination in Nigeria, 102 samples of feed (n = 30) and feed ingredients (n = 72) were collected from in-house mills of poultry farms across 12 states of Nigeria and analyzed for multiple mycotoxins using LC/MS-MS. One hundred and forty microbial metabolites were detected in the feed and feed ingredients. The most frequent mycotoxin in the feed was fumonisin B1, occurring in 97% of the samples at mean concentration of 1014 µg kg-1. AFB1 occurred in 83% of the feed samples at mean concentration of 74 µg kg-1 and in all feed ingredients except fish meal and other cereals (millet and rice). Feed samples analyzed in this study were contaminated with at least four mycotoxins: aflatoxins and fumonisin co-occurring in 80% of the samples. Peanut cake and maize contributed the most to the levels of aflatoxin and fumonisin, respectively, in the feed. Consequently, there is a need to explore other cereal- and protein-based ingredients for compounding feeds in order to reduce the risk associated with high mycotoxin (e.g. aflatoxin) intake in poultry.

Absence of Curli in Soil-Persistent Escherichia coli Is Mediated by a C-di-GMP Signaling Defect and Suggests Evidence of Biofilm-Independent Niche Specialization.

Escherichia coli is commonly viewed as a gastrointestinal commensal or pathogen although an increasing body of evidence suggests that it can persist in non-host environments as well. Curli are a major component of biofilm in many enteric bacteria including E. coli and are important for adherence to different biotic and abiotic surfaces. In this study we investigated curli production in a unique collection of soil-persistent E. coli isolates and examined the role of curli formation in environmental persistence. Although most soil-persistent E. coli were curli-positive, 10% of isolates were curli-negative (17 out of 170). Curli-producing E. coli (COB583, COB585, and BW25113) displayed significantly more attachment to quartz sand than the curli-negative strains. Long-term soil survival experiments indicated that curli production was not required for long-term survival in live soil (over 110 days), as a curli-negative mutant BW25113ΔcsgB had similar survival compared to wild type BW25113. Mutations in two genes associated with c-di-GMP metabolism, dgcE and pdeR, correlated with loss of curli in eight soil-persistent strains, although this did not significantly impair their survival in soil compared to curli-positive strains. Overall, the data indicate that curli-deficient and biofilm-defective strains, that also have a defect in attachment to quartz sand, are able to reside in soil for long periods of time thus pointing to the possibility that niches may exist in the soil that can support long-term survival independently of biofilm formation.

Traditionally Processed Beverages in Africa: A Review of the Mycotoxin Occurrence Patterns and Exposure Assessment.

African traditional beverages are widely consumed food-grade liquids processed from single or mixed grains (mostly cereals) by simple food processing techniques, of which fermentation tops the list. These beverages are very diverse in composition and nutritional value and are specific to different cultures and countries. The grains from which home-processed traditional beverages are made across Africa are often heavily contaminated with multiple mycotoxins due to poor agricultural, handling, and storage practices that characterize the region. In the literature, there are many reports on the spectrum and quantities of mycotoxins in crops utilized in traditional beverage processing, however, few studies have analyzed mycotoxins in the beverages themselves. The available reports on mycotoxins in African traditional beverages are mainly centered on the finished products with little information on the process chain (raw material to final product), fate of the different mycotoxins during processing, and exposure estimates for consumers. Regulations targeting these local beverages are not in place despite the heavy occurrence of mycotoxins in their raw materials and the high consumption levels of the products in many homes. This paper therefore comprehensively discusses for the 1st time the available data on the wide variety of African traditional beverages, the mycotoxins that contaminate the beverages and their raw materials, exposure estimates, and possible consequent effects. Mycotoxin control options and future directions for mycotoxin research in beverage production are also highlighted.

Roles for RpoS in survival of Escherichia coli during protozoan predation and in reduced moisture conditions highlight its importance in soil environments.

The soil is a complex ecosystem where interactions between biotic and abiotic factors determine the survival and fate of microbial inhabitants of the system. Having previously shown that Escherichia coli requires the general stress response regulator, RpoS, to survive long term in soil, it was important to determine what specific conditions in this environment necessitate a functional RpoS. This study investigated the susceptibility of soil-persistent E. coli to predation by the single-celled eukaryotes Acanthamoeba polyphaga and Tetrahymena pyriformis, and the role RpoS plays in resisting this predation. Strain-specific differences were observed in the predation of E. coli strains, with soil-persistent strain COB583 being the most resistant to predation by both protozoans. RpoS and curli, proteinaceous fibres used for attachment to biotic and abiotic surfaces, increased the ability of E. coli to resist predation by A. polyphaga and T. pyriformis. Furthermore, soil moisture content impacted the survival of E. coli BW25113 but wild-type COB583 had similar survival irrespective of soil moisture content. Overall, this study confirmed that RpoS contributes to the resistance of E. coli to protozoan predation and that RpoS is crucial for the increased fitness of soil-persistent E. coli against predation and reduced moisture in soil.

Bacterial species and mycotoxin contamination associated with locust bean, melon and their fermented products in south-western Nigeria.

The microbiological safety of spontaneously fermented foods is not always guaranteed due to the undefined fermenting microbial consortium and processing materials. In this study, two commonly consumed traditional condiments (iru and ogiri) and their respective raw seeds (locust bean and melon) purchased from markets in south-western Nigeria were assessed for bacterial diversity and mycotoxin contamination using 16S rRNA gene sequencing and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Two hundred isolates obtained from the raw seeds and condiments clustered into 10 operational taxonomic units (OTUs) and spanned 3 phyla, 10 genera, 14 species and 2 sub-species. Bacillus (25%) and Staphylococcus (23.5%) dominated other genera. Potentially pathogenic species such as Alcaligenes faecalis, Bacillus anthracis, Proteus mirabilis and Staphylococcus sciuri subsp. sciuri occurred in the samples, suggesting poor hygienic practice during production and/or handling of the condiments. A total of 48 microbial metabolites including 7 mycotoxins [3-nitropropionic acid, aflatoxin B1 (AFB1), AFB2, beauvericin, citrinin, ochratoxin A and sterigmatocystin] were quantified in the food samples. Melon and ogiri had detectable aflatoxin levels whereas locust bean and iru did not; the overall mycotoxin levels in the food samples were low. There is a need to educate processors/vendors of these condiments on good hygienic and processing practices.

Determination of Survival of Wildtype and Mutant Escherichia coli in Soil.

E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival was unknown. This soil survival assay protocol was developed and used to determine the role of the general stress response regulator, RpoS, in the survival of E. coli in soil. Using this soil survival assay, we demonstrated that RpoS was important for the survival of E. coli in soil. This protocol describes the development of the soil survival assay especially the recovery of E. coli inoculated into soil and can be adapted to allow further investigations into the survival of other bacteria in soil.

Mould and mycotoxin exposure assessment of melon and bush mango seeds, two common soup thickeners consumed in Nigeria.

An examination of the mould and fungal metabolite pattern in melon and bush mango seeds locally produced in Nigeria was undertaken in order to understand the mycotoxicological risk posed to consumers of both of these important and commonly consumed soup thickeners. The variation in mycotoxin levels in graded categories of both foodstuffs were also determined. Aspergillus, Fusarium, Penicillium, Mucorales and Trichoderma were the recovered fungi from the foodstuffs with Aspergillus species dominating (melon=97.8%; bush mango=89.9%). Among the Aspergillus species identified Aspergillus section Flavi dominated (melon: 72%; bush mango: 57%) and A. flavus, A. parasiticus, A. parvisclerotigenus and A. tamarii were the recovered species. About 56% and 73% of the A. flavus isolates from melon and bush mango seed samples, respectively were aflatoxigenic. Thirty-four and 59 metabolites including notable mycotoxins were found in the melon and bush mango seeds respectively. Mean aflatoxin levels (µg/kg) in melon (aflatoxin B1 (AFB1)=37.5 and total aflatoxins=142) and bush mango seeds (AFB1=68.1 and total aflatoxins=61.7) were higher than other mycotoxins, suggesting potential higher exposure for consumer populations. Significantly (p<0.05) higher levels of mycotoxins were found in hand-peeled melon and discoloured bush mango seeds than in machine-peeled melon and non-discoloured seeds except for HT-2 and T-2 toxins which occurred conversely. All melon and bush mango seeds exceeded the 2µg/kg AFB1 limit whereas all melon and 55% of bush mango seeds exceeded the 4µg/kg total aflatoxin EU limit adopted in Nigeria. This is the first report of (1) mycotoxin co-occurrence in bush mango seeds, (2) cyclopiazonic acid, HT-2 toxin, moniliformin, mycophenolic acid, T-2 toxin and tenuazonic acid occurrence, and (3) mycotoxin exposure assessment of both foodstuffs.

Co-occurrence of aflatoxins, ochratoxin A and citrinin in "egusi" melon (Colocynthis citrullus L.) seeds consumed in Ireland and the United Kingdom.

The natural co-occurrence of aflatoxins (AFs), ochratoxin A (OTA) and citrinin (CIT) in melon seed samples obtained from retailers and households in Ireland and the United Kingdom (UK) was evaluated. AFs and OTA were determined by HPLC with fluorescence detection while CIT was analysed by HPLC-MS/MS. AFB1 was detected in all (100%) samples (mean = 9.7 µg kg(-1); range = 0.2-66.5 µg kg(-1)). Mean total AFs was 12.0 µg kg(-1) (range = 0.3-82 µg kg(-1)). Commercially retailed samples showed a significantly higher AFB1 contamination (p < 0.05) than the household samples. OTA occurred in 3 (13.6%) samples, while 4 (18.2%) were contaminated with CIT at very low levels. In this study, 68% of the melon seed samples were contaminated above the 2 µg kg(-1) EU limit for AFB1 in oilseeds. These results highlight the need for the development of strategies to reduce AF contamination in "egusi" for human consumption.

The General Stress Response Is Conserved in Long-Term Soil-Persistent Strains of Escherichia coli.

UNLABELLED: Although Escherichia coli is generally considered to be predominantly a commensal of the gastrointestinal tract, a number of recent studies suggest that it is also capable of long-term survival and growth in environments outside the host. As the extraintestinal physical and chemical conditions are often different from those within the host, it is possible that distinct genetic adaptations may be required to enable this transition. Several studies have shown a trade-off between growth and stress resistance in nutrient-poor environments, with lesions in the rpoS locus, which encodes the stress sigma factor RpoS (σ(S)). In this study, we investigated a unique collection of long-term soil-persistent E. coli isolates to determine whether the RpoS-controlled general stress response is altered during adaptation to a nutrient-poor extraintestinal environment. The sequence of the rpoS locus was found to be highly conserved in these isolates, and no nonsense or frameshift mutations were detected. Known RpoS-dependent phenotypes, including glycogen synthesis and γ-aminobutyrate production, were found to be conserved in all strains. All strains expressed the full-length RpoS protein, which was fully functional using the RpoS-dependent promoter reporter fusion PgadX::gfp RpoS was shown to be essential for long-term soil survival of E. coli, since mutants lacking rpoS lost viability rapidly in soil survival assays. Thus, despite some phenotypic heterogeneity, the soil-persistent strains all retained a fully functional RpoS-regulated general stress response, which we interpret to indicate that the stresses encountered in soil provide a strong selective pressure for maintaining stress resistance, despite limited nutrient availability. IMPORTANCE: Escherichia coli has been, and continues to be, used as an important indicator species reflecting potential fecal contamination events in the environment. However, recent studies have questioned the validity of this, since E. coli has been found to be capable of long-term colonization of soils. This study investigated whether long-term soil-persistent E. coli strains have evolved altered stress resistance characteristics. In particular, the study investigated whether the main regulator of genes involved in stress protection, the sigma factor RpoS, has been altered in the soil-persistent strains. The results show that RpoS stress protection is fully conserved in soil-persistent strains of E. coli They also show that loss of the rpoS gene dramatically reduces the ability of this organism to survive in a soil environment. Overall, the results indicate that soil represents a stressful environment for E. coli, and their survival in it requires that they deploy a full stress protection response.

Correlation between aflatoxin M1 content of breast milk, dietary exposure to aflatoxin B1 and socioeconomic status of lactating mothers in Ogun State, Nigeria.

Aflatoxin M1 (AF M1), a hydroxylated metabolite of AF B1, is an important toxin that can contaminate the milk of lactating mothers. A correlation study was conducted to determine the relationship between AF M1 content of breast milk, dietary exposure to AF B1 and socioeconomic status of lactating mothers in the three Senatorial districts of Ogun State, Nigeria. Equal amounts of breast milk (20 ml) and food rations (40 kg) obtained from 50 volunteer lactating mothers and eighty-two frequently consumed food commodities in the preceding month were used for the study. The level of contamination of the foods by AF B1 was low (0.16-0.33 µg/kg) and differed significantly (p<0.05) across the state but did not exceed the EU limit of 2 µg/kg. The occurrence level of AF B1 was however high (93.75-94.45%) and was more pronounced in Ogun East Senatorial district (94.45%). Eighty-two percent of the breast milk was contaminated with AF M1 (3.49-35 ng/l) and 16% exceeded the EU limit of 25 ng/l while a 100% occurrence risk was recorded in Ogun Central Senatorial district. The socioeconomic status of the mothers also significantly influenced their dietary exposure and exposure risk of the sucklings to AF M1.