- Volume 5, Issue 9, 2023
Volume 5, Issue 9, 2023
- Research Articles
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Design and validation of a PCR screen for γ-butyrolactone-like regulatory systems in Streptomyces
More Lessγ-butyrolactone and related signalling systems are found in Streptomyces and other actinobacteria where they control the production of secondary or specialized metabolites such as antibiotics. Genetic manipulation of these regulatory systems therefore leads to changes in the secondary metabolite profile of a strain and has been used to activate previously silent secondary metabolite gene clusters. However, there is no easy way to assess the presence of γ-butyrolactone-like systems in Streptomyces strains without whole-genome sequencing. We have therefore developed and tested a PCR screen that is able to detect homologues of the commonly co-located butenolide synthase and γ-butyrolactone receptor genes. This PCR screen could be employed for the screening of strain libraries to detect signalling systems without the necessity for whole-genome sequencing.
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One year experience of Achromobacter bacteremia at a tertiary care hospital in Northern India
Introduction. Achromobacter is a Gram-negative, motile, obligate aerobic and non-fermentative bacterium. It is an emerging pathogen in the hospital environment as it is frequently found in various solutions.
Hypothesis/Gap Statement. Information about the incidence and risk factors of Achromobacter bacteremia from India is limited.
Aim. We conducted this study to identify the risk factors and underlying conditions predisposing to bacteremia by Achromobacter spp. and analyse the antibiotic resistance pattern of the isolates.
Methodology. We performed a retrospective observational study where automated blood cultures positive for Achromobacter spp. were assessed for clinical characteristics and antibiotic susceptibility patterns from January 2022 to December 2022 in the microbiology laboratory of a tertiary care centre in Northern India.
Results. A total of 14 cases (14/2435, 0.57 %) of Achromobacter spp. were identified from bloodstream infections in one year. The mean age of the patients was 37.59±23.17 years with a male predominance (8/14, 57.1 %). All patients were managed on intravenous antibiotics and intravenous access as peripheral line catheters and only 5(5/14, 35.7 %) patients were managed on central line catheters. The isolates were found highly susceptible to ticarcillin-clavulanic acid (14/14, 100.0 %) followed by fluoroquinolones (12/14, 85.72 %) and trimethoprim-sulphamethoxazole (12/14, 85.72 %). Only 57.14 % (8/14, 57.14 %) of the patients were susceptible to piperacillin-tazobactam. The all-cause 40 day mortality was observed in 35.7 % (5/14, 35.7 %) with two deaths that were directly attributable to sepsis.
Conclusion. This study provides insight into the incidence of Achromobacter bacteremia at our centre and the necessary antibiotic therapy to combat it.
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Prevalence and epidemiological characteristics of methicillin-resistant Staphylococcus aureus isolated from cattle in Bangalore India as a part of the One Health approach
In India, limited studies are available on the epidemiological aspects of methicillin-resistant Staphylococcus aureus (MRSA) infections in both animal and human settings. Herein, we investigated the prevalence, antimicrobial resistance profile and molecular characteristics of MRSA isolates recovered from cattle using the One Health approach. Out of 66 mecA-positive staphylococci, species-specific multiplex PCR detected 24 % (n=16) of isolates as MRSA. Maximum antibiotic resistance was seen against cloxacillin (94 %, n=15) and least for enrofloxacin and cephalothin (each 13 %, n=2). Overall, 13 % (n=2) of MRSA isolates were multidrug-resistant. Molecular characterization by SCCmec typing identified 88 % (n=14) of MRSA isolates as type V. Twelve isolates (75 %) belonged to novel spa-type t17242, of which 67 % (n=8) belonged to agr type I. MLST analysis revealed ST 1687 (50 %, n=8) as the most predominant sequence type. Circulation of different MRSA clones among the cattle populace offers a risk of transmission to humans through direct contact, food chain or environmental contamination. Thus, continuous monitoring of MRSA strains is imperative for early diagnosis and for establishing effective treatment strategies to restrain the disease burden caused by MRSA infections.
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The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria
Dormant bacterial cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation-promoting factors (Rpfs) are proteins that act as signalling molecules mediating growth and replication. In this study we aimed to test the effect of Rpfs from Micrococcus luteus on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro-N-acetylmuramic acid (1,6-anhydro-MurNAc). The rpf gene from Micrococcus luteus was amplified and cloned into a pET21b expression vector and the protein was expressed in Escherichia coli BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-Anhydro-MurNAc was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils was examined. Colony-forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony-forming units and of 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and in particular certain clades of the phyla Actinomycetota and Bacillota . This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.
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Monitoring and tracking the spread of SARS-CoV-2 in Asturias, Spain
Jose Maria Gonzalez-Alba, Susana Rojo-Alba, Zulema Perez-Martinez, Jose A. Boga, Marta Elena Alvarez-Arguelles, Juan Gomez, Pablo Herrero, Isabel Costales, Luz Maria Alba, Gabriel Martin-Rodriguez, Rainer Campo, Cristian Castelló-Abietar, Marta Sandoval, Fátima Abreu-Salinas, Eliecer Coto, Mercedes Rodriguez, Pablo Rubianes, Maria Luisa Sanchez, Fernando Vazquez, Luis Antuña, Victoria Álvarez and Santiago Melón GarcíaMutational analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can quantify the relative importance of variants over time, enable dominant mutations to be identified, and facilitate near real-time detection, comparison and tracking of evolving variants. SARS-CoV-2 in Asturias, an autonomous community of Spain with a large ageing population, and high levels of migration and tourism, was monitored and tracked from the beginning of the pandemic in February 2020 until its decline and stabilization in August 2021, and samples were characterized using whole genomic sequencing and single nucleotide polymorphisms. Data held in the GISAID database were analysed to establish patterns in the appearance and persistence of SARS-CoV-2 strains. Only 138 non-synonymous mutations occurring in more than 1 % of the population with SARS-CoV-2 were found, identifying ten major variants worldwide (seven arose before January 2021), 19 regional and one local. In Asturias only 17 different variants were found. After vaccination, no further regional major variants were found. Only half of the defined variants circulated and no new variants were generated, indicating that infection control measures such as rapid diagnosis, isolation and vaccination were efficient.
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- Short Communications
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Factors associated with cutaneous colonization of Mucormycetes in diabetic and non-diabetic individuals
More LessThis study was conducted to assess factors associated with cutaneous colonization of Mucormycetes in diabetic and non-diabetic individuals. A total of 800 swab samples from 200 participants including 100 diabetics and 100 non-diabetics were collected from four different body sites: (1) the forehead, (2) nasal cavity, (3) hands and (4) feet. Fungal isolation, fungal identification and antibiotic sensitivity tests were performed on the isolates. Overall, 12.0 % of the participants showed Mucormycetes colonization while the commonest fungal isolates were Mucor Species (Spp.). followed by Rhizopus spp. Diabetics had a 11 times higher odds of colonization compared to non-diabetics. The majority of the isolates were resistant to itraconazole; however, all isolates were sensitive to amphotericin B. A significant association was observed between profession and Mucormycetes (P=0.03) with significantly higher colonization in retired people compared to business people. Higher odds of colonization were demonstrated among older ages, lower class status and individuals with prolonged contact time with soil.
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Impact of inoculum density of Fusarium oxysporum f. sp. zingiberi on symptomatic appearances and yield of ginger (Zingiber officinale Roscoe)
More LessGinger (Zingiber officinale Roscoe) is an important horticultural crop valued for its medicinal and culinary properties. Fusarium yellows, caused by the ascomycete fungus Fusarium oxysporum f. sp. zingiberi (Foz), is a devastating soil-borne disease of ginger. It has curtailed ginger production in Australia and around the world, leading to significant economic losses. An integrated approach is required to manage soil-borne diseases such as those caused by Foz. However, little is known about the influence of Foz inoculum on disease severity. This study aimed to establish a minimum threshold level of spores per gram of soil required for plant infection and to develop and evaluate a pot inoculation method for screening large numbers of plants in a controlled environment. To achieve this, the dominant Australian ginger cultivar Canton was inoculated with 101, 103, 105, 106 and 107 microconidia g−1 soil. The inoculum density was positively associated with leaf and stem yellows, and rhizome discolouration, and negatively associated with root length and rhizome weight. The lowest threshold required for plant infection was 101 microconidia g−1 soil, which may provide an important basis for outbreaks of Foz in the field. This finding adds significantly to our knowledge of the impact of soil health on ginger production, thereby contributing to the integrated management of Foz. When used at a high dose, this method can facilitate reliable and accurate screening of Foz-susceptible ginger genotypes in a controlled environment.
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Antiviral activity of salt-coated materials against SARS-CoV-2
The SARS-CoV-2 pandemic demonstrated the importance of human coronaviruses and the need to develop materials to prevent the spread of emergent respiratory viruses. Coating of surfaces with antiviral materials is a major interest in controlling spread of viruses, especially in high-risk or high-traffic areas. A number of different coatings for surfaces have been proposed, each with their own advantages and disadvantages. Here we show that simple salt coating on a range of surfaces, including a novel biomass aerogel can reduce the infectivity of SARS-CoV-2 placed onto the surface. This suggests that a simple to apply coating could be applied to a range of materials and have an antiviral effect against SARS-CoV-2, as well as other potential emerging viruses.
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- Methods
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Searching for Strep A in the clinical environment during a human challenge trial: a sub-study protocol
Streptococcus pyogenes (also known as group A Streptococcus , Strep A) is an obligate human pathogen with significant global morbidity and mortality. Transmission is believed to occur primarily between individuals via respiratory droplets, but knowledge about other potential sources of transmission via aerosols or the environment is limited. Such knowledge is required to design optimal interventions to control transmission, particularly in endemic settings. We aim to detail an experimental methodology to assess the transmission potential of Strep A in a clinical environment. We will examine potential sources of transmission in up to 20 participants recruited to the Controlled human infection for penicillin against Streptococcus pyogenes (CHIPS) Trial. Three approaches to understanding transmission will be used: the use of selective agar settle plates to capture possible droplet or airborne spread of Strep A; measurement of the possible distance of Strep A droplet spread during conversation; and environmental swabbing of personal and common high-touch items to detect the presence of Strep A on hard and soft surfaces. All methods are designed to allow for an assessment of transmission potential by symptomatic, asymptomatic and non-cases. Ethical approval has been obtained through Bellberry Human Research Ethics Committee (approval 2021-03-295). Trial registration number: ACTRN12621000751875. Any results elicited from these experiments will be of benefit to the scientific literature in improving our knowledge of opportunities to prevent Strep A transmission as a direct component of the primordial prevention of rheumatic fever. Findings will be reported at local, national and international conferences and in peer-reviewed journals.
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- Pedagogy
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Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli
Synthetic biology enables the creative combination of engineering and molecular biology for exploration of fundamental aspects of biological phenomena. However, there are limited resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. We developed unigems, a set of ten plasmids that enable classroom-based investigation of gene-expression control and biological logic gates to facilitate teaching synthetic biology and genetic engineering. It is built on a high-copy plasmid backbone and is easily extensible thanks to a common primer set that facilitates Gibson assembly of PCR-generated or synthesized DNA parts into the target vector. It includes two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The set can readily be employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.
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Reflections on the Teaching Symposium at the Microbiology Society Annual Conference 2023
The Microbiology Society Education and Outreach Network (EON) recently hosted the Teaching Symposium at the Microbiology Society Annual Conference, sponsored by Access Microbiology. The presence of the Symposium as an established parallel session within the wider Annual Conference reflects the importance of high-quality, contemporary microbiology education and outreach delivered in an enthusiastic and inclusive manner. At the 2023 Symposium, a variety of pedagogical research projects in higher education learning, teaching and assessment, as well as public engagement projects, were showcased through invited talks, offered talks, flash talks and posters. The event was attended by up to 70 delegates. Several themes were noted throughout the day: engaging with Gen Z (Generation Z, those born between 1996 and 2010), active learning, art in science and engaging with non-higher education (HE) audiences. Inclusivity was a key driver in the organization of the Symposium; the room was set up to encourage discussion and participants could ask questions using an online platform as well as speaking in the room. We now encourage all speakers to consider publishing their work as a peer-reviewed article for further dissemination and impact.
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- Case Reports
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A man with culture sterile cavitating lung lesions: molecular techniques can provide the key to diagnosis
More LessA 20-year-old male presented to the Emergency Department with pyrexia, dyspnoea, chest pain and haemoptysis. Cavitating lung lesions were noted on chest X-ray and the patient was admitted to the intensive care unit where he was intubated and ventilated. Routine investigations including serial cultures did not provide an aetiological diagnosis. As such, a CT-guided lung biopsy was carried out and 16S rDNA PCR was undertaken on the sample. This identified Fusobacterium necrophorum as the causative organism. The patient was treated for Lemierre’s syndrome and successfully discharged from hospital. This case highlights how DNA tissue typing on a lung biopsy sample can be the key to successful diagnosis in an atypical pneumonia and raises the question as to whether this infrequently used approach should be added to forthcoming community acquired pneumonia guidelines.
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A rare and fatal case of tubo-ovarian abscess due to Burkholderia pseudomallei presenting as puerperal sepsis
Introduction. Melioidosis is an emerging life-threatening infectious disease caused by the Gram-negative Burkholderia pseudomallei found in contaminated soil and surface ground water. It often presents with varied clinical manifestations and has a high mortality if left untreated due to lack of clinical suspicion. Here we report a rare and fatal case of tubo-ovarian abscess presenting as puerperal sepsis due to B. pseudomallei in a young woman.
Case report. A 25-year-old female presented for medical consultation at day 43 postpartum complaining of fever on and off for the past 40 days associated with vomiting. On per speculum examination, greenish discharge from the cervix was seen and a right adnexal mass was felt which was cystic in consistency, non-tender and pushing the cervix to the left side. An exploratory laparotomy was carried out and pus was drained from below the rectus sheath, and from the right tubo-ovarian mass. Peripheral blood and the pus samples collected intraoperatively grew B. pseudomallei . The patient died on the fifth post-operative day due to septic shock with disseminated intravascular coagulation secondary to puerperal sepsis.
Conclusion. Melioidosis is a fatal but treatable disease when it is promptly diagnosed. In countries such as India, where tuberculosis is highly endemic, underdiagnosis of melioidosis can be common. Clinicians and microbiologists should have a high index of suspicion of melioidosis especially in individuals with underlying illness.
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Erosive balanitis caused by Staphylococcus haemolyticus in a healthy, circumcised adult male
More LessIntroduction. Balanitis is an inflammation of the glans penis. Balanoposthitis involves both the glans penis and prepuce and occurs only in uncircumcised males. Recurrent balanoposthitis represents a strong indication for circumcision. After Candida infections, aerobic bacteria are the second most common aetiological cause of acute infectious balanoposthitis, mainly streptococci groups B and D, and staphylococci, usually S. aureus . Their clinical manifestations are variable inflammatory changes, including diffuse erythema and oedema. Severe balanopreputial oedema with purulent exudate occurs in painful, erosive streptococcal balanoposthitis.
Coagulase-negative staphylococci (CoNS) are commensal skin bacteria, but are also recognized pathogens of the genitourinary system, mainly related to urinary tract infections. Staphylococcus haemolyticus is one of the main species of CoNS that is part of the cutaneous microflora but is also associated with nosocomial infections. In addition, S. haemolyticus also causes other infections of the male urogenital tract, such as chronic prostatitis and epididymo-orchitis, but it has not been associated with balanitis.
Case presentation. A 45-year-old man reports having suffered several episodes of balanoposthitis in the last 3 years, which were treated with topical antifungal treatments alone or associated with corticosteroids. For this reason, he underwent a postectomy by his urologist 8 months ago to avoid further recurrences.
The patient consulted for an episode of painful, erosive and exudative lesions on the glans penis and over the post-operative scars lasting 5 days. He had no urinary discomfort or inguinal lymphadenopathy. A complete blood count, biochemical analysis, C-reactive protein (CRP), prostate-specific antigen (PSA) and urinalysis were normal. Abundant growth of S. haemolyticus was obtained in the culture on tryptone soya agar with sheep blood and chocolate agar with Vitox media. The MicroScan panel CIM 37 (PM37) was used to study the antimicrobial susceptibilities of the isolated bacteria. The fungal culture on Sabouraud dextrose agar was negative. Based on the antimicrobial susceptibility study, treatment with oral ciprofloxacin and topical mupirocin was started, and the genital infection was completely cured.
Conclusion. We present a healthy, non-diabetic, circumcised male patient with severe, erosive and painful balanitis probably due to S. haemolyticus .
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Haemophagocytic lymphohistiocytosis due to Burkholderia pseudomallei in a primigravida
More LessIntroduction. Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative, saprophytic bacillus, commonly found in soil or contaminated water. As infection with this bacterium produces a wide variety of clinical manifestations the organism is aptly called the ‘great mimicker’. Even though it is non-fastidious and an easily cultivable organism, it can be misidentified in automated identification systems.
Case report. A 24-year-old primigravida presented with complaints of fever and myalgia of 45 days’ duration. She was diagnosed to have haemophagocytic lymphohistiocytosis (HLH) based on clinical and laboratory parameters. Blood and bone marrow culture sent to the microbiology laboratory grew non-fermenting Gram-negative bacilli which were misidentified as Burkholderia cepacia by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology. It was subsequently identified as B. pseudomallei by 16S rRNA gene sequencing. The patient was commenced on intensive phase therapy with intravenous ceftazidime for 2 weeks, followed by maintenance therapy with oral trimethoprim and sulfamethoxazole for 3 months. In view of HLH, she was treated with intravenous dexamethasone for 2 weeks which was later switched to oral dexamethasone for a period of 6 weeks. She responded well to the treatment, but had to undergo medical termination of her pregnancy as there was severe intrauterine growth restriction of the fetus.
Conclusion. Prognosis of melioidosis is excellent if early diagnosis and appropriate antibiotic treatment is provided. In this era of automation, it is important to determine if the suspected pathogen is listed in the database of the automated identification system.
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Case report: isolation of Hydrogenophaga from septic blood culture following near-death drowning in lakewater
A patient suffered a non-fatal wet drowning in a freshwater lake and developed bacteraemia several days later. Blood culture grew a Gram-negative rod that could not be identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 16S ribosomal RNA sequencing of the isolate identified the microbe as Hydrogenophaga laconesensis – an environmental microbe commonly found in freshwater. The recovery of multiple pathogenic micro-organisms (although not H. laconesensis ) from culture of respiratory specimens prompted the initiation of antibiotic therapy with cefepime and, later, vancomycin. The patient’s clinical course gradually improved over the course of 2 weeks and she was ultimately discharged home with minimal sequelae. To our knowledge, this is the first evidence of human infection with bacteria in the genus Hydrogenophaga . Hydrogenophaga may be considered in cases of freshwater near-drowning, and MALDI-TOF MS databases should be updated to include H. laconesensis .
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Yersinia pseudotuberculosis bacteraemia with splenic abscesses: a case report
More LessIntroduction. Yersinia pseudotuberculosis has been known to cause a variety of clinical manifestations ranging from mild enteric illness to bacteraemia with septic shock and extraintestinal abscesses. Patients with liver disease and iron overload are at risk of more severe disease manifestations.
Case Report. A middle-aged male with chronic alcohol use disorder presented with confusion and jaundice, with ascites and asterixis noted on examination. His blood work was remarkable for neutrophilic leukocytosis, elevated liver enzymes and lactate. An abdominal computed tomography scan revealed splenic microabscesses and a cirrhotic liver. Yersinia pseudotuberculosis was recovered from his blood cultures and he was treated with ceftriaxone following susceptibility results.
Conclusion. Y. pseudotuberculosis should be considered in the differential diagnosis of splenic or other extraintestinal microabscesses particularly in patients with chronic liver disease.
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Haemophagocytic lymphohistiocytosis after intravesical BCG administration for bladder cancer presenting with multiorgan failure
More LessBacillus Calmette-Guérin (BCG), is administered intravesically as an adjuvant immunotherapy for the treatment of non-muscle invasive bladder cancer. While mild non-infectious problems can occur in up to 85 % of cases, significant local and systemic complications have been reported in 1–5 % of cases. We report the case of a patient with superficial bladder cancer who developed multiorgan failure after intravesical BCG instillation including the kidney and liver with subsequent haemophagocytic lymphohistiocytosis. Our case illustrates the first reported combination of secondary haemophagocytic lymphohistiocytosis with severe renal and liver failure after BCG immunotherapy for bladder carcinoma. Treatment strategy is discussed.
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