- Volume 71, Issue 8, 2022

Rifampicin (also known as rifampin) inhibits RNA synthesis, and is used to treat tuberculosis, leprosy, staphylococcal infections and legionnaires’ disease. It can also protect at-risk populations from   Haemophilus influenzae   type b and   Neisseria meningitidis  . It is a polyketide antibiotic and is on the World Health Organization (WHO) list of essential medicines due to its critical importance to human medicine. The adverse effect of liver toxicity is controlled by testing during prolonged treatment regimes. Rifampicin’s red–orange colour can result in the colouration of sweat, tears and urine. Resistance to rifampicin arises from mutation of the target RNA polymerase or ADP ribosylation of the antibiotic or efflux. Mycobacteria may become singularly resistant to rifampicin or as part of multidrug or extensive drug resistance.

Fosfomycin (FOF) is the first antimicrobial of the epoxide class. It is commercially available in oral and parenteral formulations. Oral FOF is widely used to treat uncomplicated cystitis in women, while parenteral FOF is extensively utilized for upper urinary tract infections. FOF has a broad-spectrum bactericidal activity with a low risk of cross-resistance to other antimicrobial classes. Therefore, parenteral FOF is increasingly prescribed adjunctive therapy to treat extra-urinary tract infections caused by multidrug-resistant, Gram-negative bacteria.

Introduction. The oral cavity is one of the largest reservoirs of microorganisms and many pathogenic bacteria have been shown to be associated with the aetiology of oral cancers.

Gap Statement. Owing to the complexity of oral microbial communities and their unclear relationship with oral cancer, identification of specific bacteria which contribute to oral cancer is a key imperative.

Aim. To compare and investigate the variations in the composition of the bacterial microbiome and its functions between patients with oral tumorous lesions and healthy subjects.

Methodology. Twenty-seven samples from individuals with oral tumours (five oral benign tumours and 22 oral squamous cell carcinomas) and 15 samples from healthy subjects were collected. Genomic DNA was extracted and the V3–V5 region of the 16S rRNA gene was sequenced. Subsequently, bioinformatic assessment was conducted using QIIME2, PICRUSt and linear discriminant analysis effect size analyses (LEfSe).

Results. The oral microbiota was composed mainly of the phyla Proteobacteria (31.76 %, 35.00 %), Bacteroidetes (30.13 %, 25.13 %) and Firmicutes (23.92 %, 17.07 %) in tumorous and healthy individuals, respectively. Neisseria , Prevotella , Fusobacterium , Streptococcus , Capnocytophaga , Veillonella , Haemophilus , Prevotella , Porphyromonas and Leptotrichia were the most abundant genera. Alpha diversity in the tumour group was significantly greater than that in the healthy group (P<0.05). Differential analysis of microbes between groups demonstrated a significantly higher number of Neisseria , Veillonella , Streptococcus , Leptotrichia , Lautropia , Sphingopyxis , Sphingobium , Tannerella , Actinomyces and Rothia in healthy controls compared with the tumour group. However, the genera Treponema , Micrococcus , Pseudomonas , Janthinobacterium , Parvimos, Loktanella , Staphylococcus , Acinetobacter , Catonella , Aggregatibacter and Propionibacterium were significantly higher in the tumour group. Pathways related to cancers, cell motility, environmental adaptation, metabolism and signal transduction were enhanced in the tumour group, while functions associated with immune system diseases, replication, repair and translation were significantly enhanced in the healthy group.

Conclusion. Variations in the oral microbiota and its functions showed a correlation with oral tumours. The tumour group showed an increased abundance of some multi-drug-resistant and periodontitis-related pathogens. The significantly altered microbiotas may serve as potential biomarkers or inform combination therapy for oral tumours.

Introduction. Vaginal colonization of Group B Streptococcus (GBS) is associated with preterm births and neonatal sepsis. Thus routine screening of GBS in prenatal care is recommended.

Hypothesis. Chromogenic media (carrot broth) aids in specific and rapid detection of GBS.

Aim. To investigate the efficiency of Strep B Carrot Broth for detection of GBS in high vaginal swabs from pregnant women.

Methods. In this study 201 vaginal swab samples were collected from pregnant women. Swabs were inoculated in chromogenic media (Strep B Carrot Broth). The positive and negative cultures were inoculated on blood agar and crome agar plates. The colonies were subjected to 16S rRNA sequencing and gene-specific PCR for confirmation. The Christie Atkins Munch Peterson (CAMP) and bile esculin agar (BEA) tests were used for biochemical confirmation. PCR was performed on genomic DNA isolated from uncultured vaginal swabs.

Results. It was found that 20/201 (9.9 %) vaginal swab samples were positive in the Strep B Carrot Broth and 17/20 (85 %) and 19/20 (95 %) of these samples yielded colonies on blood agar and crome agar, respectively. Of the 181 carrot broth-negative samples, 1 (0.5 %) and 38 (20.9 %) yielded colonies on blood agar and crome agar plates, respectively. However, 16 s rRNA sequencing revealed that none of the 20 carrot broth-positive cultures were GBS and had sequence similarities to Enterococcus faecalis . This was also confirmed by using gene-specific PCR and BEA positivity. Furthermore, E. faecalis was detected by PCR in DNA isolated from 57 uncultured vaginal swabs samples, GBS could only be detected by PCR in four samples.

Conclusion. Carrot broth-based culture can lead to false-positive detection due to the presence of E. faecalis . Thus GBS-positive results in carrot broth must be confirmed by the other molecular and biochemical tests before making a final diagnosis.

Introduction. Nocardia is an opportunistic pathogen that can cause significant morbidity and mortality, particularly in the immunocompromised host. Antimicrobial susceptibility profiles vary across Nocardia spp. and vary within Australia as well as worldwide. Knowledge of local susceptibility patterns is important in informing appropriate empiric antimicrobial therapy.

Gap Statement. This is the largest study to date in Australia that correlates antimicrobial susceptibility profiles with molecular identification of Nocardia species. It is the first study that examines isolates from multiple institutions across the state of Victoria, Australia.

Aim. To investigate the species distribution and antibiotic susceptibility of Nocardia spp. isolates referred to the Mycobacterial Reference Laboratory (MRL) in Victoria, Australia from 2009 to 2019.

Methodology. We conducted a retrospective review of Nocardia spp. isolates which were identified using molecular sequencing. Antimicrobial susceptibility testing was performed using standardized broth microdilution method with Sensititre RAPMYCO1 plates. Species distribution and antibiotic susceptibility profiles were analysed.

Results. In total, 414 Nocardia isolates were identified to 27 species levels, the majority originating from the respiratory tract (n=336, 81.2 %). N. nova (n=147, 35.5 %) was the most frequently isolated, followed by N. cyriacigeorgica (n=75, 18.1 %). Species distribution varied by isolate source, with N. farcinica and N. paucivorans found more commonly from sterile sites. Linezolid and amikacin had the highest proportion of susceptible isolates (100 and 99% respectively), while low susceptibility rates were detected for ceftriaxone (59 %) and imipenem (41 %). Susceptibility to trimethoprim sulfamethoxazole varied by species (0–100 %).

Conclusion. This is the largest study to date in Australia of Nocardia species distribution and antimicrobial susceptibility patterns. N. farcinica and N. paucivorans were more likely to be isolated from sterile sites, while N. brasiliensis and N. otitidiscvarium were more likely to be isolated from skin and soft tissue. First line therapeutic antimicrobial recommendations by local guidelines were not necessarily reflective of the in vitro susceptibility of Nocardia isolates in this study, with high susceptibility detected for linezolid and amikacin, but poor susceptibility demonstrated for ceftriaxone and imipenem. Profiles for trimethoprim-sulfamethoxazole varied across different Nocardia species, warranting ongoing susceptibility testing for targeted clinical use.

Sepsis is one of the leading causes of death worldwide. The rapid identification (ID) of the causative micro-organisms is crucial for the patients’ clinical outcome. MALDI-TOF MS has been widely investigated to speed up the time-to-report for ID from positive blood cultures, and many different procedures and protocols were developed, all of them attributable either to the direct separation of microbial cells from the blood cells, or to a short subculture approach. In this study, the Rapid Sepsityper workflow (MBT Sepsityper IVD Kit, Bruker Daltonics GmbH and Co. KG, Bremen, Germany) was compared to three different short subculturing methods, established into the routine practice of three different clinical microbiology laboratories. A total of N=503 routine samples were included in this study and tested in parallel with the two approaches. Results of the rapid procedures were finally compared to routine proceedings with Gram-staining and overnight subculture. Among monomicrobial samples, the Rapid Sepsityper workflow enabled overall the correct identification of 388/443 (87.6 %) micro-organisms, while the short subculturing methods of 267/435 (61.8 %). Except for the performance with Streptococcus pneumoniae , in each one of the three sites the Rapid Sepsityper workflow proved to be superior to the short subculture method, regardless of the protocol applied, and it delivered a result from 1 to 5 h earlier.

Introduction. Rapid diagnostic tests (RDTs) that can facilitate the diagnosis of a panel of tropical infectious diseases are critically needed. DPP® Fever Panel II Asia is a multiplex lateral flow immunoassay comprising antigen and IgM panels for the diagnosis of pathogens that commonly cause febrile illness in Southeast Asia.

Hypothesis/Gap Statement. Accuracy of DPP® Fever Panel II Asia has not been evaluated in clinical studies.

Aim. To evaluate the sensitivity and specificity of DPP® Fever Panel II Asia for malaria, dengue and melioidosis.

Methodology. We conducted a cohort-based case–control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon sepsis). We included 143 and 98 patients diagnosed with malaria or dengue based on a positive PCR assay and 177 patients with melioidosis based on a culture positive for Burkholderia pseudomallei . Controls included 200 patients who were blood culture-positive for Staphylococcus aureus , Escherichia coli or Klebsiella pneumoniae , and cases of the other diseases. Serum samples collected from all patients within 24 h of admission were stored and tested using the DPP® Fever Panel II Asia antigen and IgM multiplex assays. We selected cutoff values for each individual assay corresponding to a specificity of ≥95 %. When assessing diagnostic tests in combination, results were considered positive if either individual test was positive.

Results. Within the DPP® Fever Panel II Asia antigen assay, a combination of pLDH and HRPII for malaria had a sensitivity of 91 % and a specificity of 97 %. The combination of dengue NS1 antigen and dengue antibody tests had a sensitivity of 61 % and a specificity of 91 %. The B. pseudomallei CPS antigen test had a sensitivity of 27 % and a specificity of 97 %. An odds ratio of 2.34 (95 % CI 1.16–4.72, P=0.02) was observed for the association between CPS positivity and mortality among melioidosis patients.

Conclusion. The performance of the DPP® Fever Panel II Asia for diagnosis of malaria was high and that for dengue and melioidosis was relatively limited. For all three diseases, performance was comparable to that of other established RDTs. The potential operational advantages of a multiplex and quantitative point-of-care assay are substantial and warrant further investigation.

Introduction. Haemophilus influenzae is a commensal of the respiratory tract that is frequently present in cystic fibrosis (CF) patients and may cause infection. Antibiotic resistance is well described for CF strains, and virulence factors have been proposed.

Hypothesis/Gap. The genetic diversity of H. influenzae strains present in the lungs of persons with CF is largely unknown despite the fact that this organism is considered to be a pathogen in this condition. The aim was to establish the genetic diversity and susceptibility of H. influenzae strains from persons with CF, and to screen the whole genomes of these strains for the presence of antibiotic resistance determinants and proposed virulence factors.

Methods. A total of 67 strains, recovered from respiratory samples from persons with CF from the UK (n=1), Poland (n=2), Spain (n=24) and the Netherlands (n=40), were subjected to whole-genome sequencing using Illumina technology and tested for antibiotic susceptibility. Forty-nine of these strains (one per different sequence type) were analysed for encoded virulence factors and resistance determinants.

Results. The 67 strains represented 49 different sequence types. Susceptibility testing showed that all strains were susceptible to aztreonam, ciprofloxacin, imipenem and tetracycline. Susceptibility to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cefuroxime, cefixime, ceftriaxone, cefepime, meropenem, clarithromycin, co-trimoxazole and levofloxacin ranged from 70.2–98.5%. Only 6/49 strains (12.2%) harboured acquired resistance genes. Mutations associated with a ß-lactamase-negative ampicillin-resistant phenotype were present in four strains (8.2 %). The potential virulence factors, urease, haemoglobin- and haptoglobin-binding protein/carbamate kinase, and OmpP5 (OmpA), were encoded in more than half of the strains. The genes for HMW1, HMW2, H. influenzae adhesin, a IgA-specific serine endopeptidase autotransporter precursor, a TonB-dependent siderophore, an ABC-transporter ATP-binding protein, a methyltransferase, a BolA-family transcriptional regulator, glycosyltransferase Lic2B, a helix–turn–helix protein, an aspartate semialdehyde dehydrogenase and another glycosyltransferase were present in less than half of the strains.

Conclusion. The H. influenzae strains showed limited levels of resistance, with the highest being against co-trimoxazole. Sequences encoding a carbamate kinase and a haemoglobin- and haemoglobin–haptoglobin-binding-like protein, a glycosyl transferase and an urease may aid the colonization of the CF lung. The adhesins and other identified putative virulence factors did not seem to be necessary for colonization.

Introduction. Shiga toxin-producing Escherichia coli (STEC) O157:H7 has been the most clinically significant STEC serotype in the UK for over four decades. Over the last 10 years we have observed a decrease in STEC O157:H7 and an increase in non-O157 STEC serotypes, such as O145:H28.

Gap Statement. Little is known about the microbiology and epidemiology of STEC belonging to CC32 (including O145:H28) in the UK. The aim of this study was to integrate genomic data with patient information to gain a better understanding of the virulence, disease severity, epidemic risk assessment and population structure of this clinically significant clonal complex.

Methodology. Isolates of E. coli belonging to CC32 (n=309) in the archives of public health agencies in the UK and Ireland were whole-genome-sequenced, virulence-profiled and integrated with enhanced surveillance questionnaire (ESQ) data, including exposures and disease severity.

Results. Overall, diagnoses of STEC belonging to CC32 (290/309, 94 %) in the UK have increased every year since 2014. Most cases were female (61 %), and the highest proportion of cases belonged to the 0–4 age group (53/211,25 %). The frequency of symptoms of diarrhoea (92 %), abdominal pain (84 %), blood in stool (71 %) and nausea (51 %) was similar to that reported in cases of STEC O157:H7, although cases of STEC CC32 were more frequently admitted to hospital (STEC CC32 48 % vs O157:H7  34 %) and/or developed haemolytic uraemic syndrome (HUS) (STEC CC32 9 % vs O157:H7 4 %).

The majority of STEC isolates (268/290, 92 %) had the stx2a/eae virulence gene combination, most commonly associated with progression to STEC HUS. There was evidence of person-to-person transmission and small, temporally related, geographically dispersed outbreaks, characteristic of foodborne outbreaks linked to nationally distributed products.

Conclusion. We recommend more widespread use of polymerase chain reaction (PCR) for the detection of all STEC serogroups, the development of consistent strategies for the follow-up testing of PCR-positive faecal specimens, the implementation of more comprehensive and standardized collection of epidemiological data, and routine sharing of sequencing data between public health agencies worldwide.

Introduction. Pseudomonas aeruginosa causes fatal infections in immunocompromised individuals and patients with pulmonary disorders.

Gap Statement. Agricultural ecosystems are the vast reservoirs of this dreaded pathogen. However, there are limited attempts to analyse the pathogenicity of P. aeruginosa strains associated with edible plants.

Aim. This study aims to (i) elucidate the virulence attributes of P. aeruginosa strains isolated from the rhizosphere and endophytic niches of cucumber, tomato, eggplant and chili;and (ii) compare these phenotypes with that of previously characterized clinical isolates.

Methodology. Crystal-violet microtitre assay, swarm plate experiment, gravimetric quantification and sheep blood lysis were performed to estimate the biofilm formation, swarming motility, rhamnolipid production and haemolytic activity, respectively, of P. aeruginosa strains. In addition, their pathogenicity was also assessed based on their ability to antagonize plant pathogens (Xanthomonas oryzae, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum) and kill a select nematode (Caenorhabditis elegans).

Results. Nearly 80 % of the plant-associated strains produced rhamnolipid and exhibited at least one type of lytic activity (haemolysis, proteolysis and lipolysis). Almost 50 % of these strains formed significant levels of biofilm and exhibited swarming motility. The agricultural strains showed significantly higher and lower virulence against the bacterial and fungal pathogens, respectively, compared to the clinical strains. In C. elegans, a maximum of 40 and 100% mortality were induced by the agricultural and clinical strains, respectively.

Conclusion. This investigation shows that P. aeruginosa in edible plants isolated directly from the farm express virulence and pathogenicity. Furthermore, clinical and agricultural P. aeruginosa strains antagonized the tested fungal phytopathogens, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum. Thus, we recommend using these fungi as simple eukaryotic model systems to test P. aeruginosa pathogenicity.

Introduction. Streptococcus mutans is a cariogenic bacterium that causes dental caries as well as being implicated in other dental pathologies and infective endocarditis. Bacitracin is a bactericidal antibiotic that induces cell wall stress in Gram-positive bacteria.

Gap Statement. S. mutans is among the most characterized Gram-positive bacteria. However, the transcriptome and proteome of S. mutans have received less attention, and they are actually key in understanding the pathogenesis of any bacteria. In this study, we extracted the whole proteome of S. mutans grown under bacitracin stress. Such a proteome is anticipated to offer deep insights related to physiological dynamic fluctuations and, consequently, it may provide ‘proteomic signatures’ to be identified as potential targets.

Aim. The aim of the study is to explore the general stress response that S. mutans exhibits at the proteome level when cell wall stress is imposed on it.

Methodology. A sub-MIC concentration of bacitracin was added to the growth media of S. mutans followed by whole-cell protein extraction. The proteome was then subjected to high-throughput proteomics analysis, i.e. liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins obtained through LC-MS/MS underwent analyses such as gene ontology, KEGG (Kyoto Encyclopaedia of Genes and Genomes) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis, and STRING for functional annotation, pathway enrichment and protein–protein interaction (PPI) networks, respectively. These proteins were also categorized into functional classes using the PANTHER (Protein Annotation Through Evolutionary Relationship) classification system.

Result. LC-MS/MS produced data from 321 identified proteins. From these, 41 and 30 were found to be significantly over- (≥2 fold change) and underexpressed (≤0.4 fold change), respectively. In the upregulated proteins we mostly observed sortases and proteins involved in the EPS biosynthesis pathway, whereas among the downregulated proteins the majority related to glycolysis.

Conclusion. The sortase family of proteins appear to be potential targets because they regulate various virulence factors and therefore can be targeted to inhibit multiple virulence pathways simultaneously. This study offers an understanding of proteomic fluctuations in response to cell wall stress and can thus help in identifying key players mediating virulence.