- Volume 5, Issue 10, 2023

Myxobacteria produce a variety of bioactive secondary metabolites, and with a wealth of under-researched species they hold vast potential for undiscovered compounds. With the ever-increasing need for new antibiotics, the development of novel therapeutics is vitally important. Therefore, this study aimed to extract and elucidate antimicrobial metabolites from the following myxobacteria: Myxococcus xanthus CA010 and AB022; Corallococcus exiguus DSM 14696T; Myxococcus stipitatus DSM 14675T; and Corallococcus aberystwythensis AB050AT. Metabolite mixtures were extracted in acetone from XAD-16 resin incubated in liquid cultures and analysed using GC-MS. Bioactivity was identified using a growth inhibition assay against a panel of clinically relevant prey species including Gram-positive and Gram-negative bacteria and a fungus. Growth of Klebsiella pneumoniae and Enterococcus faecalis was most affected by the metabolite mixtures and the mixtures from AB022 and AB050AT were effective against the most prey. GC-MS analysis revealed metabolites with roles in the synthesis and degradation of amino acids and fatty acids, but also identified compounds A and B with a diketopiperazine (DKP) core. With previously confirmed bioactivity of compound A, it is suggested that these DKP compounds are contributing to the antimicrobial activity observed. Furthermore, many compounds could not be identified and so these unknowns present further potential for novel bioactive compounds.

Background. Nakaseomyces glabrata, formerly Candida glabrata, is an opportunistic yeast and emerging cause of human infections. The use of broth microdilution (BMD) methodologies for caspofungin (CSP) antifungal susceptibility testing (AFST) against N. glabrata is reported to be prone to high inter-laboratory variation. We aimed to compare CSP MICs of N. glabrata isolates from our institution with those obtained by the Reference Laboratory for the same isolates.

Methods. All clinically significant N. glabrata isolates from 2019 to 2021 inclusive were reviewed. AFST was performed locally using the VITEK2 system with the AST-YS08 card, while E-tests were performed at the Mycology Reference Laboratory (MRL), and agreement between these two methods was evaluated – categorical and essential.

Results. Forty-one isolates were reviewed during the study period – 30 from blood cultures, seven from intra-operative theatre specimens and four from sterile site drain fluids. Despite an essential agreement of 100 % within ±2 log2 dilutions, marked discrepancies were noted in interpretative breakpoints between assays with 17 Minor and 16 Major category errors. Categorical agreement was 19.5 %, with the VITEK2 over-estimating resistance. A Mann–Whitney U-test assessed the relationship of MICs across the AFST modalities, and a statistically significant difference was noted, P<0.01, with a higher mean rank for VITKEK2 outputs.

Conclusion. While the VITEK2 system is highly applicable, its performance for CSP AFST is unreliable and potentially results in the mis-classification of susceptible isolates as highlighted in our study. The use of VITEK2 AST-YS08 micafungin as a sentinel echinocandin should be explored and/or the evaluation of CSP-specific E-tests as utilized by the MRL. These methods appear more consistent and less prone to the variation seen with BMD for CSP.

Introduction. The increasing antibiotic resistance like the advent of carbapenem resistant Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB), and Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to to the use of toxic and older drugs like colistin for these organisms. But worldwide there is an increase in resistance even to colistin mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance. This is impeded by the unavailability of a user-friendly phenotypic methods for use in routine clinical microbiology practice. The present study attempts to evaluate two different methods – colistin broth disc elution and MIC detection by Vitek two in comparison to CLSI approved broth microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa , and Acinetobacter baumanii clinical isolates.

Methods. Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD, Colistin Broth Disc Elution (CBDE), and Vitek two methods and was interpreted as per CLSI guidelines. The MIC results of CBDE, Vitek two were compared with that of BMD and essential agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME), major error (ME) and Cohen’s kappa (CK) was calculated. The presence of any plasmid-mediated colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated in all colistin-resistant isolates by conventional polymerase chain reaction.

Results. Colistin resistance was found in 778 (12.9 %) strains among the carbapenem resistant isolates. Klebsiella pneumoniae had the highest (18.9 %) colistin resistance by the BMD method. MIC of Vitek two had sensitivity ranging from 78.2–84.8% and specificity of >92 %. There were 171 VMEs and 323 MEs by Vitek two method, much more than CLSI acceptable range. The highest percentage of errors was committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME). On the other hand, the CBDE method performed well with EA, CA, VME and ME within acceptable range for all the organisms. The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5–98.8 % for different strains with a specificity of more than 97.6 %. None of the isolated colistin resistant organisms harboured mcr plasmids.

Conclusion. As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India. A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [ 1 ]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

This study aimed to characterize the virulence factors and antimicrobial resistance of Providencia stuartii , an opportunistic pathogen that causes human infections. We examined 45 isolates of P. stuartii both genotypically and phenotypically by studying their adherence to HeLa cells, biofilm formation, cytotoxicity and antimicrobial resistance, and analysed their genomes for putative virulence and resistance genes. This study found that most isolates possessed multiple virulence genes, including fimA, mrkA, fptA, iutA, ireA and hlyA, and were cytotoxic to Vero cells. All the isolates were resistant to amoxicillin plus clavulanic acid, levofloxacin and sulfamethoxazole plus trimethoprim, and most were resistant to ceftriaxone and cefepime. All isolates harboured extended-spectrum beta-lactamase coding genes such as bla CTX-M-2 and 23/45(51.11 %) of them also harboured bla CTX-M-9. The gene KPC-2 (carbapenemase) was detected in 8/45(17.77 %) isolates. This study also found clonality among the isolates, indicating the possible spread of the pathogen among patients at the hospital. These results have significant clinical and epidemiological implications and emphasize the importance of a continued understanding of the virulence and antimicrobial resistance of this pathogen for the prevention and treatment of future infections.

The genus Burkholderia comprises Gram-negative bacteria that are metabolically complex and versatile, often thriving in hostile settings. Burkholderia pseudomallei , the causative agent of melioidosis, is a prominent member of the genus and a clinical pathogen in tropical and sub-tropical regions. This pathogen is well known for its multidrug resistance and possible bioweapon potential. There is currently no report of the pathogen from clinical specimens in Nigeria, which might be due to misdiagnosis with phenotypic assays. This study aims to explore the accuracy of the use of phenotypic assays to diagnose B. pseudomallei in Nigeria. Two hundred and seventeen clinical samples and 28 Gram-negative clinical isolates were collected and analysed using Ashdown’s selective agar and monoclonal antibody-based latex agglutination. Species-level identification was achieved using the analytical profile index (API) 20NE system. The susceptibility of the isolates to nine different antimicrobial agents was determined using the disc diffusion method. A total of seventy-four culture-positive isolates were obtained using Ashdown’s selective agar. Twenty-two of these isolates were believed to be B. pseudomallei through the monoclonal antibody-based latex agglutination test and the API 20NE system subsequently identified 14 isolates as Burkholderia . The predominant Burkholderia species was B. cepacia with an isolation rate of 30.8 % (8/26). No isolate was distinctively identified as B. pseudomallei but five isolates were strongly suspected to be B. pseudomallei with similarity indices ranging from 81.9–91.3 %. Other bacterial species with definitive identity include Aeromonas sp., Sphingomonas sp. and Pseudomonas aeruginosa . The antibiotic susceptibility results revealed an overall resistance to amoxicillin–clavullanic acid of 71.4 %, to cefepime of 33.3 %, to trimethoprim–sulfamethoxazole of 38.1 %, to piperacillin–tazobactam of 33.3 %, to imipenem of 66.7 %, to doxycycline of 57.1% and to ceftazidime of 66.7 %. The highest intermediate resistance was observed for cefepime and piperacillin–tazobactam with a value of 66.7 % each, while there was no intermediate resistance for gentamicin, colistin and imipenem. Our findings, therefore, show that phenotypic assays alone are not sufficient in the diagnosis of melioidosis. Additionally, they provide robust support for present and future decisions to expand diagnostic capability for melioidosis beyond phenotypic assays in low-resource settings.

Background. As the COVID-19 pandemic continues, efforts to better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral shedding and transmission in both unvaccinated and vaccinated populations remain critical to informing public health policies and vaccine development. The utility of using real time RT-PCR cycle threshold values (CT values) as a proxy for infectious viral litres from individuals infected with SARS-CoV-2 is yet to be fully understood. This retrospective observational cohort study compares quantitative infectious viral litres derived from a focus-forming viral titre assay with SARS-CoV-2 RT-PCR CT values in both unvaccinated and vaccinated individuals infected with the Delta strain.

Methods. Nasopharyngeal swabs positive for SARS-CoV-2 by RT-PCR with a CT value <27 collected from 26 June to 17 October 2021 at the University of Vermont Medical Center Clinical Laboratory for which vaccination records were available were included. Partially vaccinated and individuals <18 years of age were excluded. Infectious viral litres were determined using a micro-focus forming assay under BSL-3 containment.

Results. In total, 119 specimens from 22 unvaccinated and 97 vaccinated individuals met all inclusion criteria and had sufficient residual volume to undergo viral titring. A negative correlation between RT-PCR CT values and viral litres was observed in both unvaccinated and vaccinated groups. No difference in mean CT value or viral titre was detected between vaccinated and unvaccinated groups. Viral litres did not change as a function of time since vaccination.

Conclusions. Our results add to the growing body of knowledge regarding the correlation of SARS-CoV-2 RNA levels and levels of infectious virus. At similar CT values, vaccination does not appear to impact an individual’s potential infectivity when infected with the Delta variant.

Introduction. Rhino-Orbito-cerebral mycoses are not only caused by Aspergillus spp. and Zygomycetes spp. but also can be associated with other rare species such as Neurospora spp., Cladosporium spp. and Fusarium spp. Mucormycosis is associated causatively with immunocompromised states, for example patients with comorbidities such as diabetes mellitus. Clinical symptoms of coronavirus disease (COVID) and mucormycosis in tandem are critical and relentless, frequently with no life-saving treatment.

Case series. We report three patients with COVID-19 infection, who during the course of treatment developed rhino-orbital-cerebral mycosis including oral cavity involvement. Rhinocerebral mycosis along with oral cavity involvement was diagnosed by radiological investigations and preliminary screening for fungal infection (KOH mount) in all three cases. Empirical treatment was started but patients did not respond to treatment. All patients died even after debridement and maxillectomy. On culture, rare species of fungi were isolated in all three cases which, with the help of a reference laboratory, were identified as Neurospora, Cladosporium and Fusarium. Neurospora is considered nonpathogenic to humans. Cladosporium is a dematiaceous fungus found in soil in all climates, associated with disseminated or cerebral infections; and Fusarium, though considered a saprophytic colonizer of skin and respiratory mucosa along with other bacteria, is a common cause of mycotic keratitis worldwide.

Conclusion. Immune system modifications due to COVID-19 with/without other risk factors can result in fungal co-infections that prove to be fatal for the patients. It is vital to be aware that COVID-19 patients, particularly those who are critically ill, may acquire secondary fungal infections and early detection is critical.

Introduction. One of the most serious extrapulmonary type of tuberculosis that affects people under the age of 40 is brain tuberculoma. They are space-occupying masses of granulomatous tissue that result from hematogenous spread from a distant focus of tuberculous infection by Mycobacterium tuberculosis . Symptoms and radiologic features being nonspecific usually leads to misdiagnosis and mimics a variety of other infectious diseases. Anti-tubercular drugs are essential for the successful treatment of cerebral tuberculomas.

Case Report. The authors present a case report of a 52-year-old diabetic woman, who presented to the Emergency Department of a tertiary care hospital and was diagnosed with brain tuberculomas with a brain biopsy. Brain tuberculomas are rare and could be overlooked. Therefore, this is an important consideration in cases with higher suspicions, given the rapid decline in patient condition.

Conclusion. Due to their rarity, ambiguous symptoms, and radiographic characteristics, intracranial tuberculomas continue to provide a clinical challenge and must always be considered in the differential diagnosis of cerebral space occupying lesions. As CSF may not yield positivity for both CBNAAT and smear examination, a brain biopsy specimen for culture should always be kept in mind for detecting tuberculoma and initiating anti-tubercular treatment at the earliest.

Yokenella regensburgei , belonging to the order Enterobacterales , is a rare and emerging human pathogen reported to cause both superficial and invasive infections. The 13 case reports in the literature worldwide highlight blood, bone and wound infections. To our knowledge this is the first case description of Y. regensburgei causing a urinary tract infection in a 69-year-old immunocompetent patient which was isolated in two separate specimens and identified using matrix-assisted laser desorption ionization time-of-flight MS. It was found to be susceptible to most antimicrobials but resistant to penicillin, amoxicillin-clavulanate, cefoxitin and colistin. Inducible chromosomal ampC resistance was demonstrated on disc approximation testing, and blaYOC-1 class C beta-lactamase, beta lactamase superfamily and MBL fold metallo-hydrolase genes were found on whole genome sequencing.

Introduction. Aeromonas hydrophila is an opportunistic pathogen that can cause various infections, including pneumonia, in immunocompromised individuals. This case report presents a rare occurrence of ventilator-associated pneumonia (VAP) caused by Aeromonas hydrophila in an apparently non-immunocompromised patient.

Case presentation. The patient exhibited signs and symptoms of VAP and was successfully treated with intravenous ciprofloxacin. The discussion highlights the characteristics of Aeromonas species, its virulence factors, risk factors for infection, and antibiotic profile.

Conclusion. It emphasizes the need for awareness and suspicion of Aeromonas as a potential cause of VAP in ICU settings, as well as the importance of early detection and appropriate treatment for improved outcomes.

Bifidobacterium spp. are non-spore-forming Gram-positive anaerobes that are indigenous to the human gastrointestinal tract and vagina. They are believed to be non-pathogenic organisms for humans and thus are widely used as probiotics. An 83-year-old woman taking cephalexin for 4 days was diagnosed with obstructive pyelonephritis. Y-branched Gram-positive rods were found in both anaerobic and aerobic blood culture bottles, and in an anaerobic urine culture. Bifidobacterium breve was finally identified. Ceftriaxone and metronidazole were administered to the patient, and she was discharged after intermittent catheterization for dysuria. Urinary tract infection caused by Bifidobacterium spp. is believed to be rare, but it can develop in patients with underlying urological conditions. Recognition of the characteristic morphology and conducting anaerobic urine culture may help in identifying more cases of Bifidobacterium urinary tract infections.

Introduction. The Lucio phenomenon (LP) is a characteristic reaction pattern seen in patients with diffuse lepromatous leprosy (DLL). Dual infection with Mycobacterium leprae and Mycobacterium lepromatosis in DLL has been confirmed from other endemic countries but not previously documented from India. Conventionally, LP is treated with a high dose of systemic glucocorticoid (GC) and anti-leprosy treatment (ALT). Here we report a case of leprosy lymphadenitis at initial presentation in a patient with LP and DLL due to dual infection with M. leprae and M. lepromatosis who responded favourably to tofacitinib as adjuvant to ALT and systemic GC therapy.

Case report. A 20- to 30-year-old man presented with swelling over the bilateral inguinal region, pus-filled skin lesions with multiple ulcers, fever and joint pain. Post-hospitalization investigations showed the presence of anaemia, leukocytosis, and elevated acute and chronic inflammatory markers. Skin and lymph node biopsies were suggestive of LP and leprosy lymphadenitis. The presence of M. leprae and M. lepromatosis was confirmed by PCR followed by DNA sequencing of PCR amplicons from tissue. Despite anti-leprosy treatment, oral GC and thalidomide therapy, the patient continued to develop new lesions. One month after the commencement of adjuvant tofacitinib, the patient showed excellent clinical improvement with healing of all existing lesions and cessation of new LP lesions.

Conclusion. Our case confirms the presence of dual infection with M. leprae and lepromatosis in India. Lymph node involvement as an initial presentation of DLL should be considered in endemic areas. Tofacitinib may be a promising new adjuvant therapy for recalcitrant lepra reactions.

Tuberculosis poses a considerable public health problem in countries where the disease is endemic. Osteoarticular tuberculosis represents 3–5 % of all tuberculosis cases and 10–15 % of extra-pulmonary tuberculosis cases. Involvement of the foot and ankle is rarer. We report a case of osteoarticular tuberculosis of the ankle in a 71-year-old patient with type 2 diabetes and hypertension who presented to the trauma department of the Mohammed V Military Hospital with a painful swelling of the ankle. Standard X-rays and computed tomography scans of the ankle showed inflammatory involvement of the bone and joints. Antitubercular therapy was instituted. Given the context of endemicity, any atypical presentation of lingering bone lesions should raise the suspicion of an osteoarticular tuberculosis in order to ensure early therapeutic management.

Introduction. Necrotizing soft tissue infections (NSTIs) are associated with a fulminating course because of their rapid destruction of tissue planes underlying the skin. Aeromonas -associated monomicrobial NSTIs are usually associated with exposure to fresh water, particularly among agricultural workers and fish handlers. Albeit uncommon in incidence, urgent medical and surgical intervention are required once a diagnosis has been made.

Case report. A 40-year-old male patient, a known case of alcoholic liver disease, presented to the emergency department with pain and diffuse swelling of bilateral lower limbs, which quickly progressed to form blackish discolouration and blebs. Blood for preliminary haematological and biochemical investigations, as well as fluid draining from blebs, were sent for microbiological investigation. The Gram stain revealed occasional neutrophils and Gram-negative bacilli, and pure growth in aerobic culture was identified as Aeromonas jandaei by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The patient was started on empirical antimicrobials, although lesions continued to progress and he ultimately succumbed within 12 h of hospital admission.

Conclusion. As appropriate antimicrobial therapy and early surgical intervention are required for management of the same, occupational exposure and the fulminant course should raise suspicion of Aeromonas -associated infections.

Extra-intestinal infection with non-O1/non-O139 strains of Vibrio cholerae (NOVC) is rare, though bacteremia and hepatobiliary manifestations have been reported. Reduced stomach acid, or hypochlorhydria, can increase risk of V. cholerae infection. We describe a 42-year-old woman with hypochlorhydria due to untreated Helicobacter pylori infection, gastric-bypass surgery, and chronic proton pump inhibitors (PPI) exposure, who developed acute diarrhoea following raw oyster consumption. Her symptoms were attributed to rapid gastric emptying (dumping syndrome) after a negative limited stool work-up. She had persistent diarrhoea, weight loss, and after 5 months was admitted with acute cholecystitis and NOVC bacteremia, requiring cholecystectomy. This is the first reported case of NOVC bacteremia and cholecystitis in a patient with gastric bypass. This case highlights the potential for NOVC biliary carriage, the role of hypochlorhydria as a risk factor for Vibrio infection, and the importance of excluding infectious diarrhoea in patients with new onset of symptoms compatible with dumping syndrome and a relevant travel history.