Genotypic and phenotypic comparison of clinical and environmental Acinetobacter baumannii strains.

The genotypic and phenotypic characteristics and antibiotic resistance (antibiogram) profiles of clinical (n = 13) and environmental (n = 7) Acinetobacter baumannii isolates were compared. Based on the Repetitive Extragenic Palindromic Sequence-based PCR (REP-PCR) analysis, the clinical and environmental A. baumannii isolates shared low genetic relatedness (∼60%). Multilocus sequence typing (MLST, Oxford scheme) indicated that the clinical A. baumannii were assigned to three sequence types (ST231, ST945 and ST848), while the environmental A. baumannii (excluding AB 14) were categorised into the novel ST2520. The majority of the clinical (excluding AB 5, CAB 11, CAC 37) and environmental (excluding AB 14 and AB 16) A. baumannii strains were then capable of phase variation with both the translucent (71.4%; 15/21) and opaque (95.2%; 20/21) colony phenotypes detected. The clinical isolates however, exhibited significantly (p < 0.05) higher biofilm formation capabilities (OD570: 2.094 ± 0.497). Moreover, the clinical isolates exhibited significantly (p < 0.05) higher resistance to first line antibiotics, with 92.3% (12/13) characterised as extensively drug resistant (XDR), whereas environmental A. baumannii exhibited increased antibiotic susceptibility with only 57.1% (4/7) characterised as multidrug resistant (MDR). The environmental isolate AB 14 was however, characterised as XDR. In addition, only five clinical A. baumannii isolates exhibited colistin resistance (38.5%; 5/13). The current study highlighted the differences in the genotypic, phenotypic, and antibiotic resistance profiles of clinical and environmental A. baumannii. Moreover, the environmental strains were assigned to the novel ST2520, which substantiates the existence of this opportunistic pathogen in extra-hospital reservoirs.

Insights into Bdellovibrio spp. mechanisms of action and potential applications.

Recent studies investigating Bdellovibrio spp. have found that although this predator predominantly preys on Gram-negative organisms, under certain conditions (nutrient/prey limitation), it will adapt to survive and grow axenically (without prey) or in the presence of Gram-positive bacterial prey. These advances in the understanding of predatory bacteria have stimulated a renewed interest in these organisms and the potential applications of Bdellovibrio spp. to the benefit of society. Early studies primarily focused on the application of predatory bacteria as "live antibiotics" in the medical field, probiotics in aquaculture and veterinary medicine and their use in agriculture. Additionally, studies have investigated their prevalence in wastewater and environmental sources. However, comprehending that Bdellovibrio spp. may also prey on and target Gram-positive organisms, implies that these predators could specifically be applied for the bioremediation or removal of mixed bacterial communities. Recent studies have also indicated that Bdellovibrio spp. may be useful in controlling food spoilage organisms and subsequently decrease our reliance on food additives. This review will thus highlight recent developments in understanding Bdellovibrio spp. predation strategies and focus on potential new applications of these organisms for water treatment, food preservation, enhancement of industrial processes, and in combination therapies with bacteriophages and/or antibiotics to combat multi-drug resistant organisms.

Exploring the antimicrobial resistance profiles of WHO critical priority list bacterial strains.

BACKGROUND: The antimicrobial resistance of clinical, environmental and control strains of the WHO "Priority 1: Critical group" organisms, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa to various classes of antibiotics, colistin and surfactin (biosurfactant) was determined. METHODS: Acinetobacter baumannii was isolated from environmental samples and antibiotic resistance profiling was performed to classify the test organisms [A. baumannii (n = 6), P. aeruginosa (n = 5), E. coli (n = 7) and K. pneumoniae (n = 7)] as multidrug resistant (MDR) or extreme drug resistant (XDR). All the bacterial isolates (n = 25) were screened for colistin resistance and the mobilised colistin resistance (mcr) genes. Biosurfactants produced by Bacillus amyloliquefaciens ST34 were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS). The susceptibility of strains, exhibiting antibiotic and colistin resistance, to the crude surfactin extract (cell-free supernatant) was then determined. RESULTS: Antibiotic resistance profiling classified four A. baumannii (67%), one K. pneumoniae (15%) and one P. aeruginosa (20%) isolate as XDR, with one E. coli (15%) and three K. pneumoniae (43%) strains classified as MDR. Many of the isolates [A. baumannii (25%), E. coli (80%), K. pneumoniae (100%) and P. aeruginosa (100%)] exhibited colistin resistance [minimum inhibitory concentrations (MICs) ≥ 4 mg/L]; however, only one E. coli strain isolated from a clinical environment harboured the mcr-1 gene. UPLC-MS analysis then indicated that the B. amyloliquefaciens ST34 produced C13-16 surfactin analogues, which were identified as Srf1 to Srf5. The crude surfactin extract (10.00 mg/mL) retained antimicrobial activity (100%) against the MDR, XDR and colistin resistant A. baumannii, P. aeruginosa, E. coli and K. pneumoniae strains. CONCLUSION: Clinical, environmental and control strains of A. baumannii, P. aeruginosa, E. coli and K. pneumoniae exhibiting MDR and XDR profiles and colistin resistance, were susceptible to surfactin analogues, confirming that this lipopeptide shows promise for application in clinical settings.

Cryptosporidium and Giardia in Wastewater and Surface Water Environments.

and spp. are significant contributors to the global waterborne disease burden. Waterways used as sources of drinking water and for recreational activity can become contaminated through the introduction of fecal materials derived from humans and animals. Multiple studies have reported the occurence or concentrations of these pathogens in the environment. However, this information has not been comprehensively reviewed. Quantitative microbial risk assessment (QMRA) for and can be beneficial, but it often relies on the concentrations in environmental sources reported from the literature. A thorough literature review was conducted to develop an inventory of reported and concentrations in wastewater and surface water available in the literature. This information can be used to develop QMRA inputs. and (oo)cyst concentrations in untreated wastewater were up to 60,000 oocysts L and 100,000 cysts L, respectively. The maximum reported concentrations for and in surface water were 8400 oocysts L and 1000 cysts L, respectively. A summary of the factors for interpretation of concentration information including common quantification methods, survival and persistence, biofilm interactions, genotyping, and treatment removal is provided in this review. This information can help in identifying assumptions implicit in various QMRA parameters, thus providing the context and rationale to guide model formulation and application. Additionally, it can provide valuable information for water quality practitioners striving to meet the recreational water quality or treatment criteria. The goal is for the information provided in the current review to aid in developing source water protection and monitoring strategies that will minimize public health risks.

Comparative analysis of solar pasteurization versus solar disinfection for the treatment of harvested rainwater.

BACKGROUND: Numerous pathogens and opportunistic pathogens have been detected in harvested rainwater. Developing countries, in particular, require time- and cost-effective treatment strategies to improve the quality of this water source. The primary aim of the current study was thus to compare solar pasteurization (SOPAS; 70 to 79 °C; 80 to 89 °C; and ≥90 °C) to solar disinfection (SODIS; 6 and 8 hrs) for their efficiency in reducing the level of microbial contamination in harvested rainwater. The chemical quality (anions and cations) of the SOPAS and SODIS treated and untreated rainwater samples were also monitored. RESULTS: While the anion concentrations in all the samples were within drinking water guidelines, the concentrations of lead (Pb) and nickel (Ni) exceeded the guidelines in all the SOPAS samples. Additionally, the iron (Fe) concentrations in both the SODIS 6 and 8 hr samples were above the drinking water guidelines. A >99% reduction in Escherichia coli and heterotrophic bacteria counts was then obtained in the SOPAS and SODIS samples. Ethidium monoazide bromide quantitative polymerase chain reaction (EMA-qPCR) analysis revealed a 94.70% reduction in viable Legionella copy numbers in the SOPAS samples, while SODIS after 6 and 8 hrs yielded a 50.60% and 75.22% decrease, respectively. Similarly, a 99.61% reduction in viable Pseudomonas copy numbers was observed after SOPAS treatment, while SODIS after 6 and 8 hrs yielded a 47.27% and 58.31% decrease, respectively. CONCLUSION: While both the SOPAS and SODIS systems reduced the indicator counts to below the detection limit, EMA-qPCR analysis indicated that SOPAS treatment yielded a 2- and 3-log reduction in viable Legionella and Pseudomonas copy numbers, respectively. Additionally, SODIS after 8 hrs yielded a 2-log and 1-log reduction in Legionella and Pseudomonas copy numbers, respectively and could be considered as an alternative, cost-effective treatment method for harvested rainwater.

Comparing antibiotic resistance and virulence profiles of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa from environmental and clinical settings.

Antibiotic resistance and virulence profiles of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa, isolated from water sources collected in informal settlements, were compared to clinical counterparts. Cluster analysis using repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) indicated that, for each respective species, low genetic relatedness was observed between most of the clinical and environmental isolates, with only one clinical P. aeruginosa (PAO1) and one clinical K. pneumoniae (P2) exhibiting high genetic similarity to the environmental strains. Based on the antibiograms, the clinical E. faecium Ef CD1 was extensively drug resistant (XDR); all K. pneumoniae isolates (n = 12) (except K. pneumoniae ATCC 13883) were multidrug resistant (MDR), while the P. aeruginosa (n = 16) isolates exhibited higher susceptibility profiles. The tetM gene (tetracycline resistance) was identified in 47.4 % (n = 6 environmental; n = 3 clinical) of the E. faecium isolates, while the blaKPC gene (carbapenem resistance) was detected in 52.6 % (n = 7 environmental; n = 3 clinical) and 15.4 % (n = 2 environmental) of the E. faecium and K. pneumoniae isolates, respectively. The E. faecium isolates were predominantly poor biofilm formers, the K. pneumoniae isolates were moderate biofilm formers, while the P. aeruginosa isolates were strong biofilm formers. All E. faecium and K. pneumoniae isolates were gamma (γ)-haemolytic, non-gelatinase producing (E. faecium only), and non-hypermucoviscous (K. pneumoniae only), while the P. aeruginosa isolates exhibited beta (ß)-haemolysis and produced gelatinase. The fimH (type 1 fimbriae adhesion) and ugE (uridine diphosphate galacturonate 4-epimerase synthesis) virulence genes were detected in the K. pneumoniae isolates, while the P. aeruginosa isolates possessed the phzM (phenazine production) and algD (alginate biosynthesis) genes. Similarities in antibiotic resistance and virulence profiles of environmental and clinical E. faecium, K. pneumoniae, and P. aeruginosa, thus highlights the potential health risks posed by using environmental water sources for daily water needs in low-and-middle-income countries.

Benefits and Challenges of Applying Bacteriophage Biocontrol in the Consumer Water Cycle.

Bacteria (including disinfection- and antibiotic-resistant bacteria) are abundant in the consumer water cycle, where they may cause disease, and lead to biofouling and infrastructure damage in distributions systems, subsequently resulting in significant economic losses. Bacteriophages and their associated enzymes may then offer a biological control solution for application within the water sector. Lytic bacteriophages are of particular interest as biocontrol agents as their narrow host range can be exploited for the targeted removal of specific bacteria in a designated environment. Bacteriophages can also be used to improve processes such as wastewater treatment, while bacteriophage-derived enzymes can be applied to combat biofouling based on their effectiveness against preformed biofilms. However, the host range, environmental stability, bacteriophage resistance and biosafety risks are some of the factors that need to be considered prior to the large-scale application of these bacterial viruses. Characteristics of bacteriophages that highlight their potential as biocontrol agents are thus outlined in this review, as well as the potential application of bacteriophage biocontrol throughout the consumer water cycle. Additionally, the limitations of bacteriophage biocontrol and corresponding mitigation strategies are outlined, including the use of engineered bacteriophages for improved host ranges, environmental stability and the antimicrobial re-sensitisation of bacteria. Finally, the potential public and environmental risks associated with large-scale bacteriophage biocontrol application are considered, and alternative applications of bacteriophages to enhance the functioning of the consumer water cycle, including their use as water quality or treatment indicators and microbial source tracking markers, are discussed.

Risk assessment of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa in environmental water sources: Development of surrogate models for antibiotic resistance genes.

The presence of Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and the aminoglycoside resistance genes, aac(6')-Ib and aac(6')-aph(2″), was investigated in environmental water sources obtained from informal settlements in the Western Cape (South Africa). Using ethidium monoazide bromide quantitative polymerase chain reaction (EMA-qPCR) analysis, E. faecium, K. pneumoniae, and P. aeruginosa were detected in 88.9 %, 100 %, and 93.3 % of the samples (n = 45), respectively, with a significantly higher mean concentration recorded for K. pneumoniae (7.83 × 104 cells/100 mL) over the sampling period. The aac(6')-Ib gene was detected in 95.6 % (43/45) of the environmental water samples [mean concentration of 7.07 × 106 gene copies (GC)/100 mL], while the aac(6')-aph(2″) gene was detected in 100 % (n = 45) of the samples [mean concentration of 6.68 × 105 GC/100 mL]. Quantitative microbial risk assessment (QMRA) subsequently indicated that the risks posed by K. pneumoniae and P. aeruginosa were linked to intentional drinking, washing/bathing, cleaning of the home, and swimming, in the samples collected from the various sampling sites. Surrogate risk assessment models were then designed and applied for Gram-positive [aac(6')-aph(2″) gene] and Gram-negative [aac(6')-Ib gene] pathogens that may exhibit aminoglycoside resistance. The results indicated that only the Gram-negative pathogens posed a risk (>10-4) in all the samples for cleaning of the home and intentional drinking, as well as for washing laundry by hand, garden hosing, garden work, washing/bathing, accidental consumption, and swimming at the stream and marsh sites. Thus, while environmental waters may pose a health risk of exposure to pathogenic bacteria, the results obtained indicate that screening for antibiotic resistant genes, associated with multiple genera/species, could serve as a surrogate model for estimating risks with the target group under investigation.

Biological Control of Acinetobacter baumannii: In Vitro and In Vivo Activity, Limitations, and Combination Therapies.

The survival, proliferation, and epidemic spread of Acinetobacter baumannii (A. baumannii) in hospital settings is associated with several characteristics, including resistance to many commercially available antibiotics as well as the expression of multiple virulence mechanisms. This severely limits therapeutic options, with increased mortality and morbidity rates recorded worldwide. The World Health Organisation, thus, recognises A. baumannii as one of the critical pathogens that need to be prioritised for the development of new antibiotics or treatment. The current review will thus provide a brief overview of the antibiotic resistance and virulence mechanisms associated with A. baumannii's "persist and resist strategy". Thereafter, the potential of biological control agents including secondary metabolites such as biosurfactants [lipopeptides (surfactin and serrawettin) and glycolipids (rhamnolipid)] as well as predatory bacteria (Bdellovibrio bacteriovorus) and bacteriophages to directly target A. baumannii, will be discussed in terms of their in vitro and in vivo activity. In addition, limitations and corresponding mitigations strategies will be outlined, including curtailing resistance development using combination therapies, product stabilisation, and large-scale (up-scaling) production.

Prevalence of ESKAPE pathogens in the environment: Antibiotic resistance status, community-acquired infection and risk to human health.

The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens are characterised by increased levels of resistance towards multiple classes of first line and last-resort antibiotics. Although these pathogens are frequently isolated from clinical environments and are implicated in a variety of life-threatening, hospital-associated infections; antibiotic resistant ESKAPE strains have been isolated from environmental reservoirs such as surface water, wastewater, food, and soil. Literature on the persistence and subsequent health risks posed by the ESKAPE isolates in extra-hospital settings is however, limited and the current review aims to elucidate the primary reservoirs of these pathogens in the environment, their antibiotic resistance profiles, and the link to community-acquired infections. Additionally, information on the current state of research regarding health-risk assessments linked to exposure of the ESKAPE pathogens in the natural environment, is outlined.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Human Pathogenic Bacteria Detected in Rainwater: Risk Assessment and Correlation to Microbial Source Tracking Markers and Traditional Indicators.

Roof-harvested rainwater (RHRW) was investigated for the presence of the human pathogenic bacteria Mycobacterium tuberculosis (M. tuberculosis), Yersinia spp. and Listeria monocytogenes (L. monocytogenes). While Yersinia spp. were detected in 92% (n = 25) of the RHRW samples, and L. monocytogenes and M. tuberculosis were detected in 100% (n = 25) of the samples, a significantly higher mean concentration (1.4 × 103 cells/100 mL) was recorded for L. monocytogenes over the sampling period. As the identification of appropriate water quality indicators is crucial to ensure access to safe water sources, correlation of the pathogens to traditional indicator organisms [Escherichia coli (E. coli) and Enterococcus spp.] and microbial source tracking (MST) markers (Bacteroides HF183, adenovirus and Lachnospiraceae) was conducted. A significant positive correlation was then recorded for E. coli versus L. monocytogenes (r = 0.6738; p = 0.000), and Enterococcus spp. versus the Bacteroides HF183 marker (r = 0.4071; p = 0.043), while a significant negative correlation was observed for M. tuberculosis versus the Bacteroides HF183 marker (r = -0.4558; p = 0.022). Quantitative microbial risk assessment indicated that the mean annual risk of infection posed by L. monocytogenes in the RHRW samples exceeded the annual infection risk benchmark limit (1 × 10-4 infections per person per year) for intentional drinking (∼10-4). In comparison, the mean annual risk of infection posed by E. coli was exceeded for intentional drinking (∼10-1), accidental consumption (∼10-3) and cleaning of the home (∼10-3). However, while the risk posed by M. tuberculosis for the two relevant exposure scenarios [garden hosing (∼10-5) and washing laundry by hand (∼10-5)] was below the benchmark limit, the risk posed by adenovirus for garden hosing (∼10-3) and washing laundry by hand (∼10-3) exceeded the benchmark limit. Thus, while the correlation analysis confirms that traditional indicators and MST markers should be used in combination to accurately monitor the pathogen-associated risk linked to the utilisation of RHRW, the integration of QMRA offers a more site-specific approach to monitor and estimate the human health risks associated with the use of RHRW.