Buffered Charcoal Yeast Extract Research Articles

Selectively Isolation and Molecular Identification by dnaJ Gene of Legionella pneumophila Found in Various Water Sources in Dhaka, Bangladesh

Abstract BACKGROUND: Legionella spp. are the etiological agents of Legionnaires’ disease, often severe pneumonia. After the first severe outbreak of Legionella spp., it was found widely dispersed in natural water sources and humid environments and has become a concern for these sources’ water quality and safety. The fecal indicators used for water quality assessment do not predict the presence of these pathogens; thus, in the absence of targeted analysis, the risk is unobserved until the emergence of infections. There is no study on the presence of Legionella in unattended water bodies in Bangladesh, such as swimming pools, air-conditioned cooling water, and stagnant circulatory water systems. METHODS: A total of 42 water samples were collected from 12 different sites in Dhaka, Bangladesh. Acid, heat, and chlorine treatment were applied to reduce the load of non-Legionellae in water samples. Chlorine treatment was used to kill unwanted organisms in water samples. After successful pretreatment, samples were cultured on buffered charcoal yeast extract agar (BCYE) media. Optimum pH, concentrations of chlorine, and temperature were set up by extensive trial and error culturing of Legionella on BCYE media to minimize other bacterial growth and maximize Legionella growth. Isolated colonies were confirmed primarily by morphological characteristics on BCYE media. Isolates were then confirmed by polymerase chain reaction and gel electrophoresis techniques. We detected the presence of 16S rDNA for Legionella spp. and the dnaJ gene for Legionella pneumophila species identification. Antibiotic sensitivity tests (minimum inhibitory concentration) were also conducted to see if it was alarming for our community’s health. RESULTS: Most of the Legionella spp. could grow at various temperatures, such as 45°C, 50°C, and 55°C, and they could also survive at the normal levels of chlorination, such as 0.25 mg/L and 0.5 mg/L. We have found that, out of 12 different sites, 4 out of 5 (80%) isolates from cooling tower water, 1 out of 4 (25%) from the swimming pool, and 1 out of 3 (33%) household stagnant water are contaminated with Legionella. However, 3 out of 5 isolates found are L. pneumophila from cooling water sites. Furthermore, strains of Legionella spp. were resistant to many antibiotics, such as ciprofloxacin, azithromycin, and tetracycline. CONCLUSIONS: Serious and fatal Legionella spp. (L. pneumophila) infections may be transmitted because of the widespread presence of these water sources and the large number of users in the community. This threat is worsened by the survival of Legionella under various commonly used antibiotic conditions and further exacerbated by their antibiotic resistance. Health-care professionals may face great challenges due to the lack of studies about its prevalence and treatment procedures.

Water quality influences Legionella pneumophila determination

Legionellosis is a respiratory disease of public health concern. The bacterium Legionella pneumophila is the etiologic agent responsible for >90% of legionellosis cases in the United States. Legionellosis transmission primarily occurs through the inhalation or aspiration of contaminated water aerosols or droplets. Therefore, a thorough understanding of L. pneumophila detection methods and their performance in various water quality conditions is needed to develop preventive measures. Two hundred and nine potable water samples were collected from taps in buildings across the United States. L. pneumophila was determined using three culture methods: Buffered Charcoal Yeast Extract (BCYE) culture with Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) identification, Legiolert® 10- and 100-mL tests, and one molecular method: quantitative Polymerase Chain Reaction (qPCR) assay. Culture and molecular positive results were confirmed by secondary testing including MALDI-MS. Eight water quality variables were studied, including source water type, secondary disinfectant, total chlorine residual, heterotrophic bacteria, total organic carbon (TOC), pH, water hardness, cold- and hot-water lines. The eight water quality variables were segmented into 28 categories, based on scale and ranges, and method performance was evaluated in each of these categories. Additionally, a Legionella genus qPCR assay was used to determine the water quality variables that promote or hinder Legionella spp. occurrence. L. pneumophila detection frequency ranged from 2 to 22% across the methods tested. Method performance parameters of sensitivity, specificity, positive and negative predictive values, and accuracy were >94% for the qPCR method but ranged from 9 to 100% for the culture methods. Water quality influenced L. pneumophila determination by culture and qPCR methods. L. pneumophila qPCR detection frequencies positively correlated with TOC and heterotrophic bacterial counts. The source water-disinfectant combination influenced the proportion of Legionella spp. that is L. pneumophila. Water quality influences L. pneumophila determination. To accurately detect L. pneumophila, method selection should consider the water quality in addition to the purpose of testing (general environmental monitoring versus disease-associated investigations).

Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR.

Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires' disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires' disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a "viability based flow cytometry-cell sorting and qPCR" (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires' disease.

First Report of Bacterial Leaf Scald of Plum Caused by Xylella fastidiosa in Texas.

Xylella fastidiosa is the etiological agent of Plum Leaf Scald (Greco et al. 2021). The disease was first reported in Argentina (Fernandez-Valiela et al. 1954) and then Brazil and Paraguay (French et al. 1978). In the USA, Plum Leaf Scald has been reported in the Southeastern United States (Wells et al. 1981a) and California (Hernandez-Martinez et al. 2009). In August 2021, during the Stone Fruit Survey of FY2020, plum trees (Mexican variety, Prunus mexicana) with symptoms of leaf scald, were observed in a Central Texas orchard with approximately 7% of trees exhibiting symptoms. Leaf margins were asymmetrically scorched, with necrotic areas that transitioned into chlorotic and healthy green tissues. To detect the presence of the pathogen, leaf sample petioles were tested using a double-antibody sandwich (DAS) ELISA® with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) according to manufacturer's guidelines. X. fastidiosa was detected in 20 of the 35 symptomatic samples. To confirm ELISA results, total DNA was extracted from the plant samples using the Plant DNeasy® kit (Qiagen Co. Hilden, Germany) following the manufacturer's protocol. All 20 ELISA-positive samples tested positive in a X. fastidiosa-specific real time PCR assay, using the primers XF1F and XF1R and probe XF1p (Schaad et al. 2002). Moreover, the ELISA-negative samples were also negative for PCR assay. Symptomatic samples were used to isolate the pathogen. Samples were debarked, surface-sterilized and xylem fluid collected. The fluid was gently imprinted on buffered charcoal yeast extract (BCYE) (Wells et al. 1981b) or periwinkle wilt modified (PWM) agar plates (Summer et al. 2010). After 10 days of incubation, individual colonies were observed. The colonies were slightly convex, white, opalescent, mucoid, circular with entire margins and with smooth surfaces on both media plates. Isolated colonies were triple-streak single colony purified and archived. Genomic DNA was extracted from four purified isolates using the DNeasy Blood and Tissue Qiagen® Kit, to conduct conventional PCR using HL5/HL6 (Francis et al. 2006), which identified the isolates as X. fastidiosa. Using the 16S rRNA primer pair U3/U4 (James 2010), amplicons were sequenced and compared against the NCBI database using the BLASTn algorithm. Comparative sequence analysis of amplicons from the four isolates were identical and indicated that the isolates were 100% identical to X. fastidiosa subsp. multiplex RIV5 (CP064326.1) from cherry plum, and IVIA5901 (CP047134.1) from almond. The sequences of all four isolates were deposited into NCBI GenBank, with the accession numbers OM617940 (467), OM617941 (470), OM617942 (471) and OM617943 (468). To our knowledge, this is the first report of X. fastidiosa associated with plum leaf scald in Texas, extending the geographical range of this important bacterial disease, in the Southern United States. This study highlights the importance of routine scouting of agricultural settings with a view to assessing and detecting early threats from either pests or disease and implementing relevant management strategies.

Legionella bononiensis sp. nov., isolated from a hotel water distribution system in northern Italy.

Legionella-like isolates, strains 27fs60, 30fs61 and 30cs62T, were isolated from a hotel water distribution system in the Emilia-Romagna region, Italy. Isolates were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, with transitory flagella presence and able to grow at 32-37 °C (with an optimum at 32 °C) on buffered charcoal-yeast extract agar with l-cysteine, glycine-vancomycin-polymyxin B-cycloheximide agar and Wadowsky-Yee medium agar. The strains showed positive reactions for oxidase, hippurate and gelatinase and a weakly positive reaction for catalase. Based on the EUCAST cut-off, strain 30cs62T was resistant to ciprofloxacin (5 mg l-1). The mip and rpoB gene sequences of the three strains showed close matches to those of Legionella quateirensis ATCC 49507T with similarity values of 98.2 and 94.5 %, respectively. Whole genome sequencing of the three strains was performed, resulting in G+C contents of 39.0, 39.1 and 39.0 mol%, respectively. The identity percentage measured by average nucleotide identity between the three strains and their respective closest strains were: 91.32 % L. quateirensis NCTC 12376T, 91.45 % L. quateirensis ATCC 49507T and 91.45 % L. quateirensis ATCC 49507T, respectively. The digital DNA-DNA hybridization analysis demonstrated how the isolates were separated from the most related phylogenetic Legionella species (L. quateirensis ATCC 49507T, ≤40.10 % DNA-DNA relatedness). The concatenated phylogenetic tree based on 16S rRNA, mip, rpoB and rnpB genes, shows a close relationship with L. quateirensis ATCC 49507T. The results obtained confirm the status of an independent species. The name proposed for this species is Legionella bononiensis sp. nov. with 30cs62T (=ATCC TSD-262T=DSM 112526T) as the type strain.

An outbreak of Legionnaires’ disease in newborns in Serbia

ABSTRACT Legionnaires’ disease is an atypical pneumonia caused by inhaling small droplets of water containing the bacterium Legionella spp. In newborns, it is a rare event, usually associated with water births and the use of air conditioning systems or air humidifiers. A nosocomial outbreak of Legionnaires’ disease in the maternity ward of a secondary-care hospital in Arandjelovac, Serbia is described. Two male newborns were found to be infected with Legionnella pneumophila. On Days 7 and 6 of life, respectively, they were transferred to a tertiary-care hospital with signs of severe pneumonia which was radiologically confirmed. L. pneumophila was detected in tracheal secretions from both infants by RT-PCR, and its antigens were also positive in urine samples. The source of infection in the secondary-care hospital was the internal hot water heating system, and the main contributory factor to the emergence of the infection was the low temperature of the hot water which did not kill the bacteria during the available exposure time. These two cases highlight the importance of being cautious about possible Legionnaires’ disease in maternity wards with outdated or irregularly maintained internal water supply systems. The adoption of official guidelines for the control and regular maintenance of water supply systems, including the multidisciplinary cooperation of all relevant parties, forms the basis for direct monitoring for Legionella and the prevention of new outbreaks. Abbreviations: BCYE: buffered charcoal yeast extract agar; GVPC: glycine vancomycin polymyxin cycloheximide agar; LD - Legionnaires’ disease; TR-PCR: Reverse transcription polymerase chain reaction

274. Legionella bozemanii (Fluoribacter bozemanae) Brain Abscess in a Renal Transplant Recipient

BackgroundLegionnaires’ disease is a potentially fatal multi-system disease caused by Legionella species. However, extra-pulmonary Legionella disease is rare and is typically associated with Legionella species other than L. pneumophila in immunocompromised patients. MethodsWe present a 55-year-old immunocompromised male with history of living-related renal transplant secondary to IgA nephropathy (day 0) which was complicated by T-cell mediated rejection requiring anti-thymocyte globulin and elotuzumab (day 130). ResultsPatient was hospitalized on day 184 with community-acquired pneumonia and treated with piperacillin-tazobactam and azithromycin. Three weeks later (day 214), he presented with new-onset seizures and was found to have a frontal brain abscess on MRI. His clinical course and brain imaging worsened despite undergoing multiple operative drainage procedures, placement of an extra ventricular drain, and receiving broad-spectrum antimicrobials. L. bozemanii was first identified from cerebrospinal fluid (CSF) on buffered charcoal yeast extract (BCYE) agar from day 240 and was also later confirmed by 16S rRNA sequencing. Susceptibilities were unavailable due to poor organism growth. Of note, his allergy history was significant for rash with ciprofloxacin and levofloxacin. Based on the low severity of the allergic reaction and need for central nervous system penetration, moxifloxacin 400 mg intravenously every 24 hours was initiated on day 244 in addition to broad-spectrum antibiotics. Subsequent CSF cultures were positive for L. bozemanii until the CSF culture on day 250. Due to poor clinical response, azithromycin and intrathecal polymyxin B were added for salvage therapy on day 255. His neurological status continued to worsen and he eventually succumbed to his illness on day 262. 08/31/20 MRI Brain New solitary ring-enhancing lesion with significant surrounding vasogenic edema within the anterior right frontal lobe.09/23/20 MRI Brain Post-Surgical right frontal lobe with edema, persistent cerebritis, and mass effect on the lateral ventricles.Fluoribacter Bozemanae Formerly known as Legionella bozemanii, an intracellular GNR grown on BCYE.ConclusionWe present a diagnostically challenging case of L. bozemanii brain abscess in an immunocompromised patient. To our knowledge, this is the first case of culture proven L. bozemanii brain abscess in the literature. Considering the fastidious growth of the organism, fatal nature of the infection, and narrow therapeutic profile, Legionella infection should be considered in a multi-system disease in immunocompromised patients. Disclosures Wesley Kufel, PharmD, Melinta (Grant/Research Support)Merck (Grant/Research Support)Theratechnologies, Inc. (Advisor or Review Panel member)

Closing the Brief Case: “Great Pretender”—Disseminated Blastomycosis in Western North Carolina

1. Which of the following stains are not used to visualize Blastomyces spp.? a. Gomori methenamine silver (GMS) stain b. Periodic acid-Schiff (PAS) stain c. Calcofluor white with potassium hydroxide d. Ziehl-Neelsen stainAnswer: d. Gomori methenamine silver and periodic acid-Schiff are used to visualize Blastomyces during histopathology. Calcofluor white with potassium hydroxide is used to stain direct specimens. Ziehl-Neelsen is used for the identification of mycobacteria and not used for the identification of endemic fungi. 2. How is Blastomyces species infection usually acquired? a. Through cutaneous direct inoculation b. Inhalation of conidia c. Bloodstream spread after trauma d. Animal biteAnswer: b. Although direct inoculation is possible, the most common way of acquiring blastomycosis is through inhalation of the conidia. Blastomycosis after an animal bite has been described but is not common. 3. Which culture medium is preferred for recovery of Blastomyces spp.? a. Buffered charcoal yeast extract (BCYE) b. Brain heart infusion with sheep’s blood and gentamicin (BHISG) c. MacConkey agar d. Chocolate agarAnswer: b. Blastomyces spp. grow on BHISG, BCYE, and chocolate agar. However, BHISG is preferred due to the enriched growth of Blastomyces spp. and the inhibition of bacterial growth, which is particularly important for nonsterile specimens. The BCYE plate is sometimes included with fungal cultures to allow for the isolation of Nocardia and other aerobic actinomycetes, but any bacterial growth outcompetes Blastomyces spp. on this medium. The MacConkey agar is used for the recovery of enteric Gram-negative bacteria in bacterial cultures and does not support the growth of Blastomyces spp.

Legionella pneumonia as a cause of atypical pneumonia in a tertiary care hospital in Sri Lanka

Atypical pneumonia which contributes to an important proportion of community acquired pneumonia and Legionella pneumophila is a noteworthy pathogen worldwide. Legionnaires’ disease, the severe form of pneumonia is predominantly caused by Legionella pneumophila serogroup 1. This study was conducted to determine the presence of Legionella pneumophila as a pathogen and the associated risk factors among the patients admitted with atypical pneumonia to a tertiary care hospital in Sri Lanka. Seventy-five adult patients diagnosed with atypical pneumonia attending professorial medical unit of Colombo South Teaching Hospital, Sri Lanka were enrolled. Risk factors related to the disease was assessed by an interviewer administered questionnaire. Expectorated sputum was processed on supplemented buffered charcoal yeast extract medium and urine specimens were tested for Legionella pneumophila serogroup 1 antigen. Fifty-six percent of the patients were males and 91% were residing in urban areas. Smoking was admitted by 32% and that of alcohol consumption was in 25%. Composting and potting, distant travelling and frequent use of air conditioning was reported in 28%, 37% and 4% respectively. Majority of participants were not exposed to cooling towers, humidifiers or plumbing and not visited high risk areas. Among the study group, 21% had diabetes, 24% had ischaemic heart disease and 36% had chronic lung diseases. None of the sputum samples grew Legionella pneumophila, and all urine samples were negative for the antigen of Legionella pneumophila serogroup 1. It is concluded that, in this population of community acquired atypical pneumonia, Legionella pneumophila was not identified as a causative agent.

Comparison of two culture methods for the enumeration of Legionella pneumophila from potable water samples.

Legionella infections have steadily increased in the United States over the last 20 years, and most of these infections have been attributed to contaminated water. The gold standard for confirmation of Legionella presence in water is culturing with Buffered Charcoal Yeast Extract (BCYE) agar. Following many modifications, this method is still time-consuming, expensive, and can take longer than 10 days for full confirmation. The Legiolert is a newer and simpler culture product that is claimed to be able to quantify Legionella pneumophila in 7 days with high sensitivity and specificity and does not need further confirmation for the presence of L. pneumophila. This study compared the culturability of L. pneumophila occurring in a simulated home plumbing system using both Legiolert and BCYE agar methods. Out of 185 water samples, Legiolert and BCYE method detected L. pneumophila in 83 and 85% of the samples, respectively. The two methods were determined to be statistically equivalent for culturability of L. pneumophila, though the detected levels by Legiolert were slightly higher than the BCYE method. The molecular confirmation of positive (n = 254) and negative wells (n = 82) with Legiolert also showed a high specificity of 96.5% (i.e., 3.5% false positives (9/254) and 0% false negatives (0/82)).

Legionella pneumophila recovery using Legiolert and a traditional culture method

AbstractMethods for recovery and identification of Legionella spp. in water have not been improved significantly since the traditional culture method using buffered charcoal yeast extract (BCYE) agar was developed in 1979. Variations to the BCYE method include sample concentration techniques, acid and heat treatment of samples, and inclusions of antibiotics and dyes in the medium to improve Legionella recovery. A new culture method—Legiolert—was developed and is targeted specifically for recovery and enumeration of Legionella pneumophila, the pathogen responsible for >97% of Legionella pneumonia cases. This study assessed the performance of the Legiolert and BCYE methods in water samples. A total of 247 potable water samples were collected and analyzed using both methods. Mean recoveries in reagent water were similar for both methods (~70%), while Legiolert was more likely than BCYE to detect L. pneumophila when it is present in low numbers.Article Impact StatementThe Legiolert method for enumeration of Legionella pneumophila in water is a significant improvement over the buffered charcoal yeast extract method and should be adopted.

Detection of Legionella pneumophila and Legionella dumoffii Biochemically in Water Samples in Baghdad City, Iraq

Background: Legionellae can be isolated from various sources of water. Legionella pneumophila istransmitted via contaminated water and caused many diseases like pneumonia; lungs abscesses and fever.Objectives: Isolation and identification of L. pneumophila from environmental samples; study the factorsthat affect the frequency of L. pneumophila; and some oxidizing substances on their growth.Materials and Method: A total of one hundred water samples were collected from Cooling tower; Watertanks; Tap water and Swab from inner tap water. All samples were cultured on Buffered charcoal yeast extract(BCYE) agar and Buffered charcoal yeast extract (BCYE) broth to isolate Legionella pneumophila andLegionella dumoffii. Effect of pH; temperature; chlorine; Iron and effect of soluble zinc on L. pneumophilagrowth were studied.Results: The isolates gave positive results for the tests of catalase, oxidase, gelatin degradation, nitratereduction and beta-lactamase. Iron; Soluble Zinc; Chloride; Calcium hardness (CaCO3); Turbidity and silverwere ? 3ppmas; 1-3 ppmas; 0.2-0.5 ppm; 600 ppm; ? 0.3 and 3 ppmas respectively. The distribution of L.pneumophila in the water samples were studied.Conclusion: FatalL. pneumophila may be transmitted by drinking contaminated water and led to deathbecause they caused pneumonia.

Legionella antimicrobial sensitivity testing: comparison of microbroth dilution with BCYE and LASARUS solid media.

ObjectivesThere is a lack of international unification for AST methodology for Legionella pneumophila. Current literature contains multiple possible methods and this study compares each of them to determine methodological concordance.MethodsAntibiotic susceptibility of 50 L. pneumophila strains was determined using broth microdilution (BMD), serial antimicrobial dilution in traditional buffered charcoal yeast extract (BCYE) agar (as well as comparison with gradient strip overlay on BCYE) and in a novel charcoal-free agar (LASARUS) for rifampicin, azithromycin, levofloxacin and doxycycline.ResultsThe deviation of tested media relative to BMD highlighted the overall similarity of BMD and LASARUS across all antimicrobials tested (within one serial dilution). BCYE agar dilution showed an increased MIC of up to five serial dilutions relative to BMD, while MICs by gradient strip overlay on BCYE were elevated by two to three serial dilutions, with the exception of doxycycline, which was decreased by three serial dilutions relative to MIC values determined by BMD. The MIC range for azithromycin was wider than for other antimicrobials tested and found to be caused by the presence or absence of the lpeAB gene.ConclusionsBMD-based antimicrobial susceptibility testing (AST) methodology should be the internationally agreed gold standard for Legionella spp. AST, as is common for other bacterial species. Traditional BCYE gave significantly elevated MIC results and its use should be discontinued for Legionella spp., while MIC determination using LASARUS solid medium gave results concordant (within one serial dilution) with BMD for all antimicrobials tested. To the best of our knowledge, this study is the first to identify the lpeAB gene in UK isolates.

Respiratory Distress in an Adolescent Girl.

1. Bobby Kumar, MD* 2. Katie Meyer, DO* 3. Leon Rosenberg, DO* 4. Alyson Trillo, DO* 5. Ashley Van Putten, DO, PGY3* 1. *Salah Foundation Children’s Hospital, Fort Lauderdale, FL A 17-year-old girl with past medical history of iron deficiency anemia and depression presented with 6 days of fever (maximum temperature of 103.6°F), cough, congestion, and mild shortness of breath. Other symptoms included nonbloody, nonbilious emesis, decreased appetite, diarrhea, chest pain, body aches, and abdominal pain. When her shortness of breath worsened, she presented to the emergency department for evaluation. On presentation, she was febrile, with oral temperature of 103.1°F, tachycardia, and in severe respiratory distress with subcostal and supraclavicular retractions, decreased breath sounds on the bases, and significant crackles on the right side. Initial oxygen saturation was 89% on room air, with improvement to 98% on 2 L/min of oxygen via nasal cannula. Chest radiograph shows extensive right-sided infiltrates as well as a focal patchy infiltrate on the left (Fig 1). Figure 1. Anteroposterior (a) and lateral (b) chest radiographs showing extensive right-sided infiltrates with patchy infiltrates on the left. Due to right upper quadrant abdominal pain and persistent diarrhea, an abdominal ultrasound was performed, which was unremarkable. Initial laboratory testing on admission revealed no elevated white blood cell count, mild microcytic anemia (hemoglobin 11.3 g/dL, mean corpuscular volume 60.6), hyponatremia (sodium 125 mmol/L), hypokalemia (potassium 3.3 mmol/L), elevated aspartate aminotransferase (141 units/L) and alanine aminotransferase (57 units/L), elevated γ -glutamyl transferase (52 units/L), prolonged coagulation studies (prothrombin time 17.7 seconds and partial thromboplastin time 30.0 seconds), minimally elevated lipase (200 units/L), positive toxicology for cannabis, elevated C-reactive protein of 36.6 mg/dL, negative urine pregnancy test, urinalysis with 100 mg/dL of protein, and a specific gravity of 1.024. Screening for sexually transmitted infections was negative. The …

Evaluation of GVPC and BCYE Media for Legionella Detection and Enumeration in Water Samples by ISO 11731: Does Plating on BCYE Medium Really Improve Yield?

Legionella spp are the causative agents of Legionnaires’ diseases, which is a pneumonia of important public health concern. Ubiquitous freshwater and soil inhabitants can reach man-made water systems and cause illness. Legionella enumeration and quantification in water systems is crucial for risk assessment and culture examination is the gold standard method. In this study, Legionella recovery from potable water samples, at presumably a low concentration of interfering microorganisms, was compared by plating on buffered charcoal yeast extract (BCYE) and glycine, vancomycin, polymyxin B, cycloheximide (GVPC) Legionella agar media, according to the International Standard Organization (ISO) 11731: 2017. Overall, 556 potable water samples were analyzed and 151 (27.1%) were positive for Legionella. Legionella grew on both BCYE and GVPC agar plates in 85/151 (56.3%) water samples, in 65/151 (43%) on only GVPC agar plates, and in 1/151 (0.7%) on only BCYE agar plates. In addition, GVPC medium identified Legionella species other than pneumophila in six more samples as compared with the culture on BCYE. Although the medians of colony forming units per liter (CFU/L) detected on the BCYE and GVPC agar plates were 2500 and 1350, respectively (p-value < 0.0001), the difference did not exceed one logarithm, and therefore is not relevant for Legionella risk assessment. These results make questionable the need to utilize BCYE agar plates to analyze potable water samples.

Investigation of the Incidence of Legionella and Free-Living Amoebae in Swimming Pool Waters and Biofilm Specimens in Istanbul by Different Methods

Legionella bacteria living in free form or in biofilm and free-living amoebae (FLA) can infect humans through swimming pools and can cause various diseases. FLA may also threaten the health of swimmers because they are capable of being hosts for Legionella and some other bacteria. The aim of this study was to investigate the presence of total aerobic heterotrophic bacteria (TAHB), FLA and Legionella bacteria in swimming pool waters and biofilm samples in Istanbul by using culture and FISH methods. Water plate count agar (wPCA), buffered charcoal yeast extract (BCYE) agar supplemented with glycinevancomycin-polymyxin-cycloheximide (GVPC) and Escherichia coli cultivated non-nutrient agar (NNA) were used for the culture of TAHB, Legionella and FLA. For the FISH method analysis , Leg 705 and Leg PNE1 probes labeled with fluorescent dye for Legionella and ACANTHA probe for Acanthamoeba genus FLA were used. Legionella pneumophila serogroup 1 ATCC 33152, L.pneumophila serogroup 3 ATCC 33155 and Acanthamoeba castellani ATCC 50373 were used as positive controls. TAHB were grown in 92% and 84% of water and biofilm samples. Although Legionella bacteria could not be grown in any of the water samples, it was detected in 6 (24%) water samples by FISH method. Although these bacteria could be grown in 1 (4%) of biofilm samples, 7 (28%) were detected by FISH method. FLA were found to be 16% by culture in water samples and 28% by FISH analysis. These amoebae were detected 8% and 20% in biofilm samples by culture and FISH method, respectively. It was determined that one of the isolates of FLA had thermotolerant activity (potentially pathogenic). L.pneumophila serogroup 1 was detected in one water sample and in four biofilm samples. According to the culture method, TAHB and FLA were found to be more common in water samples than in biofilm samples and Legionella bacteria were more common in biofilm samples than in water samples (p≤ 0.05). In the detection of Legionella bacteria, the superiority of FISH method compared to culture method was found to be statistically significant (p≤ 0.05). In this study, it was found that the number of TAHB in the controlled swimming pools was within the limits determined by the Ministry of Health (≤ 200 cfu/ml). It will be appropriate to examine both water and biofilm samples for the investigation of TAHB, FLA and Legionella. It may be appropriate to use both culture and FISH methods to detect the presence of FLA in water and biofilm samples. This study is the first study to investigate the presence of Legionella and FLA in swimming pools in Istanbul, and further studies are needed to examine more pool water and biofilm samples. With the data obtained, the health principles and controls of swimming pools will be re-considered and will be contributed to public health.

Survey of pathogenic bacteria of biofilms in a metropolitan drinking water distribution system.

Bacteria, especially pathogenic bacteria, were detected in order to estimate the safety of drinking water distribution systems (DWDSs). Sixteen biofilms and 12 water samples (six retained and six flowing) were collected from a city DWDS in eastern China. Biofilms were observed using scanning electron microscopy. Cultivable bacteria of biofilms were counted by heterotrophic plate counts, ranging from 3.61×101 to 1.67×106 CFU·cm-2. Coliforms, Salmonella, Shigella, Vibrio and Legionella were separated by Eosin-Methylene Blue (EMB) agar, Salmonella chromogenic medium, Shigella chromogenic medium, Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar and Buffered Charcoal Yeast Extract (BCYE) agar and 13/16, 8/16, 7/16, 6/16, 0/16 biofilm samples were found to be positive, respectively. Retained and flowing water samples were collected to estimate the influence of hydrodynamic conditions on biofilm detachment. All six retained water samples were positive for bacteria, the count ranged from 1.2×103 to 2.8×104 CFU·mL-1 and 2/6, 3/6, 2/6, 0/6, 0/6 samples were positive for coliforms, Salmonella, Shigella, Legionella and Vibrio, respectively. While only three of six flowing water samples were bacteria positive, the counts ranged from 102 to 103 CFU·mL-1, 2/6 were coliform positive and no pathogens were detected under testing. The results show that there are pathogens in DWDS biofilms, which can cause health-related problems if detached from their surfaces.

Susceptibility of Legionella pneumophila to antimicrobial agents and the presence of the efflux pump LpeAB.

Legionella pneumophila strains resistant to antimicrobial agents are rare. We tested 10 antimicrobial agents against clinical and environmental strains and performed WGS to screen for the presence of resistance mechanisms. A total of 122 clinical and environmental strains of L. pneumophila collected between 2000 and 2017 and characterized by serogroup and ST were included. Antimicrobial susceptibility was tested by gradient diffusion tests on buffered charcoal yeast extract agar medium supplemented with α-ketoglutarate (BCYE-α) and a subgroup of strains were whole-genome sequenced using Illumina technology and analysed. All strains showed a WT MIC distribution for ciprofloxacin, levofloxacin, moxifloxacin, rifampicin, cefotaxime, tetracycline and trimethoprim/sulfamethoxazole. All strains of L. pneumophila serogroup 1, ST1 (18/122; 14.7%) showed reduced susceptibility to azithromycin (MIC 0.5-1 mg/L) and harboured the efflux pump component lpeAB. Two strains of L. pneumophila serogroup 5 (ST1328) and one strain of serogroup 4 (ST1973) also showed reduced susceptibility to azithromycin (MIC 0.5 mg/L). They harboured lpeAB gene variants with 91.37% and 92.52% nucleotide identity, respectively, compared with the lpeAB genes of serogroup 1, ST1 strains. Our collection of L. pneumophila strains was susceptible to most antimicrobial agents except azithromycin. Gradient diffusion tests on BCYE-α test medium detected strains with reduced susceptibility to azithromycin. All L. pneumophila serogroup 1, ST1 strains showed reduced susceptibility to macrolides and contained the efflux pump component lpeAB. Reduced susceptibility to azithromycin in non-serogroup 1 strains may be due to the presence of an lpeAB efflux pump variant.

Isolation and Identification of Legionella spp. in environmental water sources based on macrophage infectivity potentiator (mip) gene sequencing in southwest Iran.

Legionella species are widespread in natural water sources and man-made aqueous environments, as well as fresh-water. The present study was conducted owing to the lack of research regarding the prevalence of Legionella spp in the water sources of Ahvaz city in southwest Iran. In this study the macrophage infectivity potentiator (mip) gene sequencing was used for identification of various Legionella species isolated from different water sources. In this study, 144 water samples were collected and inoculated on the buffered charcoal-yeast extract (BCYE) agar and modified Wadowsky-Yee (MWY) medium. The DNA was extracted from positive cultures. The Legionella species were confirmed by amplifying a 654 bp fragment of the 16S rRNA gene. The mip gene of all isolates were amplified by PCR and purified for sequencing. The mip gene sequences were analyzed by jPHYDIT software version 1. The results showed a 13.9% (20/144) prevalence of Legionella spp. in water sources of Ahvaz city, southwest Iran. Analyzing of the mip gene sequences showed, out of 20 Legionella isolates, 13 isolates (54.1%) were positive for L. pneumophila, 5 isolates (20.8%) were positive for L. worsleinsis, one isolates for each one of L. dumoffi and L. fairfieldensis, (4.1%). According to our research, the occurrence of Legionella spp in water sources could be a hazard for the health systems especially in the hospitals. The regular monitoring of these water sources by health planners may therefore be useful for decreasing the risk for Legionella spp. infections.

Environmental surveillance of Legionella pneumophila in distal water supplies of a hospital for early identification & prevention of hospital-acquired legionellosis.

Background & objectives:Legionella pneumophila, a ubiquitous aquatic organism is found to be associated with the development of the community as well as hospital-acquired pneumonia. Diagnosing Legionella infection is difficult unless supplemented with, diagnostic laboratory testing and established evidence for its presence in the hospital environment. Hence, the present study was undertaken to screen the hospital water supplies for the presence of L. pneumophila to show its presence in the hospital environment further facilitating early diagnosis and prevention of hospital-acquired legionellosis.Methods:Water samples and swabs from the inner side of the same water taps were collected from 30 distal water outlets present in patient care areas of a tertiary care hospital. The filtrate obtained from water samples as well as swabs were inoculated directly and after acid buffer treatment on plain and selective (with polymyxin B, cycloheximide and vancomycin) buffered charcoal yeast extract medium. The colonies grown were identified using standard methods and confirmed for L. pneumophila by latex agglutination test.Results:About 6.66 per cent (2/30) distal water outlets sampled were found to be contaminated with L. pneumophila serotype 2-15. Isolation was better with swabs compared to water samples.Interpretation & conclusions:The study showed the presence of L. pneumophila colonization of hospital water outlets at low levels. Periodic water sampling and active clinical surveillance in positive areas may be done to substantiate the evidence, to confirm or reject its role as a potential nosocomial pathogen in hospital environment.