Environmental Isolates Research Articles

Phenotypical Comparison between Environmental and Clinical Acinetobacter baumannii Strains Isolated from an Intensive Care Unit.

Acinetobacter baumannii (A. baumannii) causes a variety of nosocomial infections that mainly affect critically ill patients in intensive care units (ICUs). The objective of this study was to assess the prevalence of A. baumannii in the ICU environment and evaluate the antibiotic resistance and biofilm formation ability of the environmental isolates compared to those isolated from ICU patients simultaneously. A total of 166 non-duplicate ICU samples (80 environmental and 86 clinical) were collected between January 2019 and January 2020. Antimicrobial susceptibility detection was determined using the disc diffusion method, and the strains were evaluated for the minimum inhibitory concentration (MIC) of imipenem (IMP) using broth microdilution or metallo-β-lactamase (MBL) detection according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The isolates' capacity to produce biofilms was evaluated using the tube method and the crystal violet microtitre plate-based method. A. baumannii was identified in 25 (31.25%) environmental and 30 (34.88%) clinical samples, and beds were the most infected (60%). Both types of isolate demonstrated a rate surpassing 80% resistance to the tested antibiotics. Phenotypically, the environmental and clinical strains were found to be MBL producers. Fourteen environmental (56%) and 15 clinical (50%) strains were found to be moderate biofilm producers, indicating that each isolate has a high biofilm-forming capacity. These results show that the spread of multidrug-resistant (MDR) A. baumannii in an ICU setting emphasises the necessity of disinfecting and cleaning medical devices and surfaces to prevent and restrict cross-transmission. Intensive surveillance and infection control methods are also of paramount importance.

Comparative genomic analysis of a multidrug-resistant ExPEC strain IITR156 isolated from the confluence of three Indian rivers

Extra intestinal pathogenic Escherichia coli (ExPEC) with the potential to cause a wide range of infections in extra-intestinal sites is emerging threat to human health. Earlier studies have explored genomic characteristics of ExPEC isolates of human, avian, and porcine origin but genomic studies of environmental ExPEC isolates are very scarce. The environmental isolate, Escherichia coli IITR156 isolated in this study from the Triveni Sangam water sample showed resistance to antibiotics belonging to different classes including penicillin, beta-lactam combination agent, macrolide, cephalosporin, fluoroquinolone and aminoglycoside. Strain IITR156 was found to be harbouring plentitude of antibiotic resistant genes (ARGs) such as blaOXA-1, blaTEM-1, blaCTX-M-15, aac(3)-IIc and aac(6′)-Ib-cr, virulence genes including chuA, chuS, ybtA, ybtE, ybtP, epcA, and epcB, heavy metal resistant genes (HMRGs) such as arsRBC, cusB, cusF, cutA, tehA, nikABC, rcnA, rcnB, zraP, zitB, and zupT . Many of the ARGs and virulence genes were carried by mobile genetic elements such as genomic islands, transposons and plasmids indicating horizontal acquisition of genes. Comparative genomic analysis revealed that the strain IITR156 is closely related to other E. coli with ST 8881 isolated from human clinical samples. The presence of clinically relevant ARGs and virulence genes in an environmental isolate and its close relatedness to clinical isolates raise concerns and warrant immediate public attention.

Enzyme Production and Inhibitory Potential of Pseudomonas aeruginosa: Contrasting Clinical and Environmental Isolates.

(1) Background: This study summarizes the findings of two studies investigating the inhibitory effects of Pseudomonas aeruginosa strains from clinical and environmental sources against gram-positive and gram-negative bacteria and fungi. The studies also analyzed the correlation between enzyme production and inhibitory effects to gain insights into the antimicrobial capabilities of P. aeruginosa strains; (2) Methods: Both studies employed similar methodologies, including the use of disk diffusion and well diffusion methods to assess the inhibitory effects of P. aeruginosa strains against target pathogens. Enzyme production was analyzed through various biochemical assays to determine the diversity and frequencies of enzyme secretion among the strains; (3) Results: A comparative analysis of enzyme production in P. aeruginosa strains from clinical sources revealed significant variations in enzyme production, with hemolysin and protease being the most commonly produced enzymes. Gelatinase production showed lower rates, whereas chondroitinase and hyaluronidase were absent or occurred less frequently. In contrast, a comparative analysis of enzyme production in environmental isolates showed different patterns, indicating adaptation to environmental conditions. Pyocyanin production was absent in all environmental isolates. The inhibitory effects against gram-positive and gram-negative bacteria varied among different P. aeruginosa strains, with strain-specific variations observed. Limited inhibitory effects were observed against fungi, primarily toward gram-positive bacteria; (4) Conclusions: The findings highlight the strain-specific nature of inhibitory effects and enzyme production in P. aeruginosa strains. The correlation between enzyme production and inhibitory effects against gram-positive bacteria suggest a potential role of specific enzymes, such as hemolysin and protease, in the antimicrobial activity. The complexity of the relationship between enzyme production and the inhibition of different pathogens requires further investigation. The results emphasize the potential of P. aeruginosa strains as sources for antimicrobial strategies, particularly against gram-positive bacteria. Future research should focus on understanding the mechanisms underlying these inhibitory effects and exploring their therapeutic applications.

Spectrometric Characterization of Clinical and Environmental Isolates of Aspergillus Series Versicolores.

Aspergillus series Versicolores are molds distributed among 17 species, commonly found in our environment, and responsible for infections. Since 2022, a new taxonomy has grouped them into 4 major lineages: A. versicolor, A. subversicolor, A. sydowii, and A. creber. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) could be a faster and more cost-effective alternative to molecular techniques for identifying them by developing a local database. To evaluate this technique, 30 isolates from Aspergillus series Versicolores were used. A total of 59 main spectra profiles (MSPs) were created in the local database. This protocol enabled accurate identification of 100% of the extracted isolates, of which 97% (29/30) were correctly identified with a log score ≥ 2.00. Some MSPs recorded as Aspergillus versicolor in the supplier's database could lead to false identifications as they did not match with the correct lineages. Although the local database is still limited in the number and diversity of species of Aspergillus series Versicolores, it is sufficiently effective for correct lineage identification according to the latest taxonomic revision, and better than the MALDI-TOF MS supplier's database. This technology could improve the speed and accuracy of routine fungal identification for these species.

High Prevalence of Plasmid-Mediated Quinolone Resistance among ESBL/AmpC-Producing Enterobacterales from Free-Living Birds in Poland.

In this study, we investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) in extended-spectrum β-lactamase- (ESBL) and/or AmpC-type β-lactamase-producing Enterobacterales isolates from free-living birds in Poland. The prevalence of the qnrB19 gene was 63%, and the distribution of isolates in terms of bacterial species was as follows: 67% (22/33) corresponded to Escherichia coli, 83% (5/6) to Rahnella aquatilis, 44% (4/9) to Enterobacter cloacae and 33% (1/3) to Klebsiella pneumoniae. The qnrB19 gene was also found in a single isolate of Citrobacter freundii. The molecular characteristics of qnrB19-positive isolates pointed to extended-spectrum beta lactamase CTX-M as the most prevalent one (89%) followed by TEM (47%), AmpC (37%) and SHV (16%). This study demonstrates the widespread occurrence of PMQR-positive and ESBL/AmpC-producing Enterobacterales isolates in fecal samples from wild birds. In this work, plasmid pAM1 isolated from Escherichia coli strain SN25556 was completely sequenced. This plasmid is 3191 nucleotides long and carries the qnrB19 gene, which mediates decreased susceptibility to quinolones. It shares extensive homology with other previously described small qnrB19-harboring plasmids. The nucleotide sequence of pAM1 showed a variable region flanked by an oriT locus and a Xer recombination site. The presence of a putative recombination site was detected, suggesting that interplasmid recombination events might have played a role in the development of pAM1. Our results highlight the broad geographical spread of ColE-type Qnr resistance plasmids in clinical and environmental isolates of Enterobacterales. As expected from the results of phenotypic susceptibility testing, no resistance genes other than qnrB19 were identified.

Complete genome sequence of mcr-9 containing Leclercia adecarboxylata.

Here, we provide the genome sequence of a Leclercia adecarboxylata isolated from a screen of an environmental bacterial isolate library for resistance to the plant flavonoid berberine. We detected the colistin resistance gene mcr-9, located on an IncFII(pECLA) plasmid.

Genomic dissection of endemic carbapenem resistance reveals metallo-beta-lactamase dissemination through clonal, plasmid and integron transfer

Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish endemicity remains limited. We analysed two decades of blaIMP-4 evolution in a hospital using sequence data from 270 clinical and environmental isolates (including 169 completed genomes) and identified the blaIMP-4 gene across 7 Gram-negative genera, 68 bacterial strains and 7 distinct plasmid types. We showed how an initial multi-species outbreak of conserved IncC plasmids (95 genomes across 37 strains) allowed endemicity to be established through the ability of blaIMP-4 to disseminate in successful strain-genetic setting pairs we termed propagators, in particular Serratia marcescens and Enterobacter hormaechei. From this reservoir, blaIMP-4 persisted through diversification of genetic settings that resulted from transfer of blaIMP-4 plasmids between bacterial hosts and of the integron carrying blaIMP-4 between plasmids. Our findings provide a framework for understanding endemicity and spread of MBLs and may have broader applicability to other carbapenemase-producing organisms.

Current Understanding of Potential Linkages between Biocide Tolerance and Antibiotic Cross-Resistance.

Antimicrobials (e.g., antibiotics and biocides) are invaluable chemicals used to control microbes in numerous contexts. Because of the simultaneous use of antibiotics and biocides, questions have arisen as to whether environments commonly treated with biocides (e.g., hospitals, food processing, wastewater, agriculture, etc.) could act as a reservoir for the development of antibiotic cross-resistance. Theoretically, cross-resistance could occur if the mechanism of bacterial tolerance to biocides also resulted in antibiotic resistance. On the other hand, biocides would likely present a higher evolutionary barrier to the development of resistance given the different modes of action between biocides and antibiotics and the broad-based physicochemical effects associated with most biocides. Published studies have shown that the induction of biocide tolerance in a laboratory can result in cross-resistance to some antibiotics, most commonly hypothesized to be due to efflux pump upregulation. However, testing of environmental isolates for biocide tolerance and antibiotic cross-resistance has yielded conflicting results, potentially due to the lack of standardized testing. In this review, we aim to describe the state of the science on the potential linkage between biocide tolerance and antibiotic cross-resistance. Questions still remain about whether the directed evolution of biocide tolerance and the associated antibiotic cross-resistance in a laboratory are or are not representative of real-world settings. Thus, research should continue to generate informative data to guide policies and preserve these tools' utility and availability.

Characterizing the type 6 secretion system (T6SS) of E. cloacae SBP-8 and its role in pathogenesis and bacterial competition

Despite the relevance of E. cloacae as an opportunistic pathogen, very little is known about its pathogenicity mechanism and the factors influencing its virulence. The mechanism of E. cloacae pathogenicity appears to be complex and multifactorial, with the presence of different putative virulence factors whose role is still not clear in the development of the disease. In this study, we systematically investigated the role of T6SS (type six secretion system) of E. cloacae SBP-8, an environmental isolate, in eukaryotic and bacterial cell interaction. Analysis of the genome sequence of E. cloacae SBP-8 revealed the presence of sets of genes coding for the expression of one complete T6SS cluster, which is similar to T6SS-1 cluster of E. cloacae ATCC 13047 (clinical isolates). In addition, an Hcp effector protein was detected in the secretome, and this secretion depended on ClpV, an Atpase of T6SS, confirming that strain SBP-8 produces functional T6SS. Deletion of T6SS-associated gene clpV did not induce any significant change in the life span and rate of colonization in C. elegans. No major significant change was observed in the expression profiling of antimicrobial genes (clec-60, clec-85, clec-87 and lys-1) and toll-like receptor (toll-1) gene, involved in stimulating an immune response against the pathogen. No difference in the ability to invade and proliferate in intestinal cells and phagocytosis by macrophages was observed. In addition, we demonstrated that the ability of E. cloacae SBP-8 to out-compete Escherichia coli was reliant upon its T6SS in contact-dependent manner. Our results show that T6SS of the environmental isolates is required for interbacterial competition but not for invasion and proliferation inside host cells.

Antimicrobial Resistance Patterns and Risk Factors Associated with ESBL-Producing and MDR Escherichia coli in Hospital and Environmental Settings in Lusaka, Zambia: Implications for One Health, Antimicrobial Stewardship and Surveillance Systems.

Antimicrobial resistance (AMR) is a public health problem threatening human, animal, and environmental safety. This study assessed the AMR profiles and risk factors associated with Escherichia coli in hospital and environmental settings in Lusaka, Zambia. This cross-sectional study was conducted from April 2022 to August 2022 using 980 samples collected from clinical and environmental settings. Antimicrobial susceptibility testing was conducted using BD PhoenixTM 100. The data were analysed using SPSS version 26.0. Of the 980 samples, 51% were from environmental sources. Overall, 64.5% of the samples tested positive for E. coli, of which 52.5% were from clinical sources. Additionally, 31.8% were ESBL, of which 70.1% were clinical isolates. Of the 632 isolates, 48.3% were MDR. Most clinical isolates were resistant to ampicillin (83.4%), sulfamethoxazole/trimethoprim (73.8%), and ciprofloxacin (65.7%) while all environmental isolates were resistant to sulfamethoxazole/trimethoprim (100%) and some were resistant to levofloxacin (30.6%). The drivers of MDR in the tested isolates included pus (AOR = 4.6, CI: 1.9-11.3), male sex (AOR = 2.1, CI: 1.2-3.9), and water (AOR = 2.6, CI: 1.2-5.8). This study found that E. coli isolates were resistant to common antibiotics used in humans. The presence of MDR isolates is a public health concern and calls for vigorous infection prevention measures and surveillance to reduce AMR and its burdens.

A novel Queuovirinae lineage of Pseudomonas aeruginosa phages encode dPreQ0 DNA modifications with a single GA motif that provide restriction and CRISPR Cas9 protection in vitro.

Deazaguanine DNA modifications are widespread in phages, particularly in those with pathogenic hosts. Pseudomonas phage iggy substitutes ∼16.5% of its genomic 2'-deoxyguanosine (G) with dPreQ0, and the iggy deazaguanine transglycosylase (DpdA) is unique in having a strict GA target motif, not observed previously. The iggy PreQ0 modification is shown to provide protection against both restriction endonucleases and Cas9 (when present in PAM), thus expanding our understanding of the deazaguanine modification system, its potential, and diversity. Phage iggy represents a new genus of Pseudomonas phages within the Queuovirinae subfamily; which have very little in common with other published phage genomes in terms of nucleotide similarity (<10%) and common proteins (<2%). Interestingly, shared similarity is concentrated in dpdA and preQ0 biosynthesis genes. TEM imaging confirmed a siphovirus morphology with a prolate icosahedral head and a non-contractile flexible tail with one long central tail spike. The observed protective effect of the deazaguanine modification on the iggy DNA may contribute to its broad within-species host range. Phage iggy was isolated on Pseudomonas aeruginosa PAO1, but also infects PDO300, PAK, PA14, as well as 10 of 27 tested environmental isolates and 13 of 20 tested clinical isolates of P. aeruginosa from patients with cystic fibrosis.

Genome Sequence of Lichtheimia ornata, an Emerging Opportunistic Mucorales Pathogen.

Lichtheimia ornata is an emerging opportunistic Mucorales pathogen that is associated with fatal infections in immunocompromised individuals. While these environmentally acquired infections have rarely been reported to date, cases were noted in a recent analysis of coronavirus disease 2019 (COVID-19)-associated mucormycosis in India. Here, we report the annotated genome sequence of the environmental isolate CBS 291.66.

Molecular characterization and in-depth genome analysis of Enterobacter sp. S-16.

Enterobacter species are considered to be an opportunistic human pathogen owing to the existence of antibiotic-resistant strains and drug resides; however, the detailed analysis of the antibiotic resistance and virulence features in environmental isolates is poorly characterized. Here, in the study, we characterized the biochemical characteristics, and genome, pan-genome, and comparative genome analyses of an environmental isolate Enterobacter sp. S-16. The strain was identified as Enterobacter spp. by using 16S rRNA gene sequencing. To unravel genomic features, whole genome of Enterobacter sp. S-16 was sequenced using a hybrid assembly approach and genome assembly was performed using the Unicycler tool. The assembled genome contained the single conting size 5.3 Mbp, GC content 55.43%, and 4500 protein-coding genes. The genome analysis revealed the various gene clusters associated with virulence, antibiotic resistance, type VI secretion system (T6SS), and many stress tolerant genes, which may provide important insight for adapting to changing environment conditions. Moreover, different metabolic pathways were identified that potentially contribute to environmental survival. Various hydrolytic enzymes and motility functions equipped the strain S-16 as an active colonizer. The genome analysis confirms the presence of carbohydrate-active enzymes (CAZymes), and non-enzymatic carbohydrate-binding modules (CBMs) involved in the hydrolysis of complex carbohydrate polymers. Moreover, the pan-genome analysis provides detailed information about the core genes and shared genes with the closest related Enterobacter species. The present study is the first report showing the presence of YdhE/NorM in Enterobacter spp. Thus, the elucidation of genome sequencing will increase our understanding of the pathogenic nature of environmental isolate, supporting the One Health Concept.

Acinetobacter baumannii from grass: novel but non-resistant clones.

Acinetobacter baumannii is one the most worrisome nosocomial pathogens, which has long been considered almost mainly as a hospital-associated bacterium. There have been some studies about animal and environmental isolates over the last decade. However, little effort has been made to determine if this pathogen dwells in the grass. Here, we aim to determine the evolutionary relationships and antibiotic resistance of clones of A. baumannii sampled from grass to the major human international clones and animal clones. Two hundred and forty genomes were considered in total from four different sources for this study. Our core and accessory genomic epidemiology analyses showed that grass isolates cluster in seven groups well differentiated from one another and from the major human and animal isolates. Furthermore, we found new sequence types under both multilocus sequence typing schemes: two under the Pasteur scheme and seven for the Oxford scheme. The grass isolates contained fewer antibiotic-resistance genes and were not resistant to the antibiotics tested. Our results demonstrate that these novel clones appear to have limited antibiotic resistance potential. Given our findings, we propose that genomic epidemiology and surveillance of A. baumannii should go beyond the hospital settings and consider the environment in an explicit One Health approach.

Impact of a new hospital with close to 100% single-occupancy rooms on environmental contamination and incidence of vancomycin-resistant Enterococcus faecium colonization or infection: a genomic surveillance study

Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of nosocomial infection, driven by its ability to spread between patients and persist in the hospital environment. To investigate the impact of a long-established cardiothoracic hospital moving to new premises with close to 100% single-occupancy rooms on the rates of environmentalcontamination and infection or colonization by VRE. Prospective environmental surveillance for VRE was conducted at five time-points between April and November 2019, once in the original building, and four times in the new building. Incidence rate ratios (IRRs) of VRE infection/colonization were determined for the one-year period before and after the hospital move, and compared to a nearby hospital. In the original location, the first environmental screen found 29% VRE positivity. The following four screens in the new location showed a significant reduction in positivity (1-6%; P<0.0001). The VRE infection/colonization rates were halved in the new location (IRR: 0.56; 95% confidence interval: 0.38-0.84), compared to the original location,contrasting with an increase in a nearby hospital (1.62; 1.17-2.27) over the same time-period. Genomic analysis of the environmental isolates was consistent with reduced transmission in the new hospital. The use of single-occupancy rooms was associated with reducedenvironmental contamination with VRE, and lower transmission and isolation of VRE from clinical samples. The cost-effectiveness of single-occupancy room hospitals in reducing healthcare-associated infections should be reassessed in the context of operational costs of emerging pandemic and increasing antimicrobial resistance threats.

Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung.

The genetic disease cystic fibrosis (CF) frequently leads to chronic lung infections by bacteria and fungi. We identified three individuals with CF with persistent lung infections dominated by Clavispora (Candida) lusitaniae. Whole-genome sequencing analysis of multiple isolates from each infection found evidence for selection for mutants in the gene MRS4 in all three distinct lung-associated populations. In each population, we found one or two unfixed, non-synonymous mutations in MRS4 relative to the reference allele found in multiple environmental and clinical isolates including the type strain. Genetic and phenotypic analyses found that all evolved alleles led to loss of function (LOF) of Mrs4, a mitochondrial iron transporter. RNA-seq analyses found that Mrs4 variants with decreased activity led to increased expression of genes involved in iron acquisition mechanisms in both low iron and replete iron conditions. Furthermore, surface iron reductase activity and intracellular iron were much higher in strains with Mrs4 LOF variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatitidis infection also had a non-synonymous LOF mutation in MRS4. Together, these data suggest that MRS4 mutations may be beneficial during chronic CF lung infections in diverse fungi, perhaps, for the purposes of adaptation to an iron-restricted environment with chronic infections. IMPORTANCE The identification of MRS4 mutations in Clavispora (Candida) lusitaniae and Exophiala dermatitidis in individuals with cystic fibrosis (CF) highlights a possible adaptive mechanism for fungi during chronic CF lung infections. The findings of this study suggest that loss of function of the mitochondrial iron transporter Mrs4 can lead to increased activity of iron acquisition mechanisms, which may be advantageous for fungi in iron-restricted environments during chronic infections. This study provides valuable information for researchers working toward a better understanding of the pathogenesis of chronic lung infections and more effective therapies to treat them.

Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30°C, 37°C, and 40°C) and osmotolerance (0.5M, 1M, and 1.5M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format.

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes (e.g., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5-2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.

“Relationship between antibiotic resistance, biofilm formation, virulence factors and source of origin of Pseudomonas aeruginosa environmental isolates with regard to the presence of metallo-β-lactamase-encoding genes”

A total of 557 water samples were evaluated and of these, 23 were positive for the presence of Pseudomonas aeruginosa. Approximately 91.7% of them were weak biofilm formers. Only 4 isolates showed antimicrobial resistance. All isolates presented Twitching motility, a positive result for the production of pyocyanin, alkaline protease, and hemolysins. The genotypic tests showed: lasA, (95.6%) lasB (95.6%), exoS (95.6%), exoT (91.3%), toxA (91.3%), akgO (91.3%), plcN (91.3%) aprA (86.9%), phzM (78.3%), and pvdA (60.9%). For genes encoding metallo-beta-lactamase, it was found: blaVIM (56.6%), blaSPM (4.3%), and blaSIM (47.8%). A strong association was found between the metallo-beta-lactamase producing genes, nine genes of virulence factors and the motility (r = 0.6231). The very close clonal profile suggests a probable similarity between the isolates from different cities. Thus, P. aeruginosa can be present in water supplies with variable virulence capacities and can generate a huge concern for human, animal, and environmental health.

Biofilm analyses and exoproduct release by clinical and environmental isolates of Burkholderia pseudomallei from Brazil

Objective: To characterize biofilm production by clinical (n=21) and environmental (n=11) isolates of Burkholderia pseudomallei and evaluate the production of proteases, hemolysins and siderophores. Methods: Initially, the 32 strains were evaluated for biofilm production in Müller-Hinton broth-1% glucose (MH-1% glucose) and BHI broth-1% glucose, using the crystal violet staining technique. Subsequently, growing (48 h) and mature (72 h) biofilms were evaluated by confocal microscopy. Finally, the production of proteases, hemolysins and siderophores by planktonic aggregates, growing biofilms and mature biofilms was evaluated. Results: All isolates produced biofilms, but clinical isolates had significantly higher biomass in both MH-1% glucose (P&lt;0.001) and BHI-glucose 1% (P=0.005). The structural analyses by confocal microscopy showed thick biofilms, composed of multiple layers of cells, homogeneously arranged, with mature biofilms of clinical isolates presenting higher biomass (P=0.019) and thickness of the entire area (P=0.029), and lower roughness coefficient (P=0.007) than those of environmental isolates. Protease production by growing biofilms was significantly greater than that of planktonic (P&lt;0.001) and mature biofilms (P&lt;0.001). Hemolysin release by planktonic aggregates was higher than that of biofilms (P&lt;0.001). Regarding siderophores, mature biofilms presented higher production than growing biofilms (P&lt;0.001) and planktonic aggregates (P&lt;0.001). Conclusions: Clinical isolates have higher production of biofilms than their environmental counterparts; protease and siderophores seem important for growth and maintenance of Burkholderia pseudomallei biofilms.