- Volume 68, Issue 10, 2019

Respiratory tract infections are responsible for over 2.8 million deaths per year worldwide. Colonization is the first step in the process of microbes occupying the respiratory tract, which may lead to subsequent infection. Carriage, in contrast, is defined as the occupation of microbial species in the respiratory tract. The duration of carriage may be affected by host immunity, the composition and interactions between members of the microbial community, and the characteristics of colonizing bacteria, including physiology associated with being present in a bacterial biofilm. Numerous vaccines have been implemented to control infections caused by bacteria that can colonize and be subsequently carried. Such vaccines are often species-specific and may target a limited number of strains thereby creating a vacant niche in the upper respiratory tract. Epidemiological changes of bacteria found in both carriage and disease have therefore been widely reported, since the vacant niche is filled by other strains or species. In this review, we discuss the use of carriage-prevalence studies in vaccine evaluation and argue that such studies are essential for (1) examining the epidemiology of carriage before and after the introduction of new vaccines, (2) understanding the dynamics of the respiratory tract flora and (3) identifying the disease potential of emerging strains. In an era of increasing antibiotic resistance, bacterial carriage-prevalence studies are essential for monitoring the impact of vaccination programmes.

Coxiella burnetii is an obligate intracellular pathogen that causes the zoonotic disease Q fever in humans, which can occur in either an acute or a chronic form with serious complications. The bacterium has a wide host range, including unicellular organisms, invertebrates, birds and mammals, with livestock representing the most significant reservoir for human infections. Cell culture models have been used to decipher the intracellular lifestyle of C. burnetii , and several infection models, including invertebrates, rodents and non-human primates, are being used to investigate host–pathogen interactions and to identify bacterial virulence factors and vaccine candidates. However, none of the models replicate all aspects of human disease. Furthermore, it is becoming evident that C. burnetii isolates belonging to different lineages exhibit differences in their virulence in these models. Here, we compare the advantages and disadvantages of commonly used infection models and summarize currently available data for lineage-specific virulence.

Introduction. Combretum leprosum (Combretaceae) is commonly found in the Northeast Region of Brazil and is known for several bioactivities, including antimicrobial ones. Because of increasing bacterial antibiotic resistance, natural products from several plants have been studied as putative adjuvants to antibiotic activity, including products from C. leprosum.

Aims. This study was carried out to investigate the structural properties, bactericidal activity and antibiotic modifying action of the lupane triterpene 3β,6β,16β-trihydroxylup-20(29)-ene (CLF1) isolated from C. leprosum Mart. leaves.

Methods. The CLF1 was evaluated by the Fourier transform infrared spectroscopy method and the antibacterial activity of this compound was assayed alone and in association with antibiotics by microdilution assay.

Results. Spectroscopic studies confirmed the molecular structure of the CLF1 and permitted assignment of the main infrared bands of this natural product. Microbiological assays showed that this lupane triterpene possesses antibacterial action with clinical relevance against Staphylococcus aureus . The CLF1 triterpene increased antimicrobial activity against the multidrug-resistant Escherichia coli 06 strain when associated with the antibiotics gentamicin and amikacin. Synergistic effects were observed against the S. aureus 10 strain in the presence of the CLF1 triterpene with the antibiotic gentamicin.

Conclusion. In conclusion, the CLF1 compound may be useful in the development of antibacterial drugs against the aforementioned bacteria.

Introduction. Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae , Corynebacterium ulcerans and Corynebacterium pseudotuberculosis . For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al. J . Med . Microbiol. 2016;65(12):1521–1527).

Aims. We aimed to improve the quadruplex method by adding a 16S rRNA gene target as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays, thus avoiding the possibility of false-negative reporting.

Methodology. Universal 16S rRNA gene primers and a probe were defined. The novel method was tested using 36 bacterial isolates and 17 clinical samples. Experimental robustness to temperature and reagent concentration variations was assessed.

Results. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species Corynebacterium belfantii) from C. ulcerans and C. pseudotuberculosis . Complete diagnostic specificity, sensitivity and experimental robustness were demonstrated. The lower limit of detection for C. diphtheriae , C. ulcerans and tox targets was 1.86 genome copies per 5 µl reaction volume. The method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England – National Infection Service, London, UK).

Conclusion. This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

Purpose. To identify potential biomarkers and/or therapeutic targets for bladder cancer we characterized and analysed the composition of the urinary microbiota from bladder cancer and non-cancer patients.

Methodology. In this study, we collected urine samples from 29 bladder cancer patients and 26 non-cancer patients. To avoid contamination and the impact of antibiotics, urine specimens were collected in a clean manner prior to antibiotic administration. Using the amplicon-based next-generation sequencing approach, the potential determinant bacteria were estimated in a between-group comparison. The results illustrated the differences in microbiota abundance among cancer and non-cancer patients and the overall number of cases carrying these bacteria.

Results. We found that the urine samples contained a conserved microbiota with four phyla ( Firmicutes , Actinobacteria , Proteobacteria and Bacteroidetes ), which accounted for 94.4 % of bacteria in all cases. Comparing the microbiota between the bladder cancer and control group, five genera of bacteria ( Streptococcus , Bifidobacterium , Lactobacillus , Veillonella and Actinomyces ) existed in all samples, but with significant intergroup differences (P<0.05). The bladder cancer patients presented with a higher abundance of Actinomyces , while the other strains were enriched in the control group. A higher abundance of Actinomyces europaeus was also observed in the bladder cancer group compared to the control group.

Conclusion. The samples collected from the bladder cancer patients displayed a significantly different pattern relative to those from the control group. The higher abundance of A. europaeus observed in bladder cancer patient samples also suggests that the strain may be indicative of bladder cancer. The urinary microbiota may be a potential biomarker and therapeutic target for bladder cancer.

Introduction. The remarkable intrinsic resistance of Fusarium species to most antifungal agents results in high mortality rates in the immunocompromised population.

Aims. This study aimed to investigate the epidemiology, clinical features and antifungal susceptibility of Fusarium isolates in patients with invasive fusariosis.

Methodology. A total of 27 patients admitted to a referral hospital from January 2008 to June 2017 were evaluated. Antifungal susceptibility testing of isolates was performed by broth microdilution according to the Clinical and Laboratory Standards Institute guidelines.

Results. Haematological malignancy was the predominant underlying condition, with an incidence of invasive fusariosis of 14.8 cases per 1000 patients with acute lymphoid leukaemia and 13.1 cases per 1000 patients with acute myeloid leukaemia. The Fusarium solani species complex (FSSC) was the most frequent agent group, followed by the Fusarium oxysporum species complex (FOSC). Voriconazole showed the best activity against Fusarium, followed by amphotericin B. Itraconazole showed high minimum inhibitory concentration values, indicating in vitro resistance. Clinical FSSC isolates were significantly (P<0.05) more resistant to amphotericin B and voriconazole than FOSC isolates.

Conclusion. The present antifungal susceptibility profiles indicate a high incidence of fusariosis in patients with haematological malignancy. Species- and strain-specific differences in antifungal susceptibility exist within Fusarium in this setting.

Aim. The aim of this work was to characterize the response of Candida albicans to atorvastatin, and to assess its in vivo antifungal capability.

Methodology. The effect of atorvastatin on the growth and viability of C. albicans was assessed. The ability of the statin to alter cell permeability was quantified by measuring amino acid and protein leakage. The response of C. albicans to atorvastatin was assessed using label-free quantitative proteomics. The in vivo antifungal activity of atorvastatin was assessed using Galleria mellonella larvae infected with C. albicans.

Results. Atorvastatin inhibited the growth of C. albicans. The atorvastatin-treated cells showed lower ergosterol levels than the controls, demonstrated increased calcofluor staining and released elevated quantities of amino acids and protein. Larvae infected with C. albicans showed a survival rate of 18.1±4.2 % at 144 h. In contrast, larvae administered atorvastatin (9.09 mg kg−1) displayed a survival rate of 60.2±6.4 % (P<0.05). Label-free quantitative proteomics identified 1575 proteins with 2 or more peptides and 465 proteins were differentially abundant (P<0.05). There was an increase in the abundance of enzymes with oxidoreductase and hydrolase activity in atorvastatin-treated cells, and squalene monooxygenase (4.52-fold increase) and lanosterol synthase (2.84-fold increase) were increased in abundance. Proteins such as small heat shock protein 21 (−6.33-fold) and glutathione peroxidase (−2.05-fold) were reduced in abundance.

Conclusion. The results presented here indicate that atorvastatin inhibits the growth of C. albicans and is capable of increasing the survival of G. mellonella larvae infected with C. albicans.

Introduction. Among beta-haemolytic streptococci, Streptococcus pyogenes (GAS) is the prototype agent of bacterial pharyngitis and causes other human infections. Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes GAS-like infections, while Streptococcus agalactiae (GBS) is a common neonate pathogen that is rarely associated with pharyngitis.

Aim. To determine the prevalence and persistence of beta-haemolytic streptococci throat carriage and type the bacterial population.

Methods. Throat swabs were collected from 121 children and 127 young adult volunteers and cultured. Colonized volunteers were screened quarterly, for up to 1 year, while beta-haemolytic streptococci could be detected. Isolates were identified and submitted to antimicrobial susceptibility testing and epidemiological typing.

Results. Carriage was detected in 34 (13.7 %) volunteers. Seventeen children carried GAS (14 %), while 17 young adults carried SDSE (8, 6.3 %), GBS (4, 3.1 %), GAS (3, 2.4 %) and the Streptococcus anginosus group (2, 1.6 %). Persistent carriage was detected for up to 6 months in two children and for up to 1 year in three young adults. Three new emm subtypes were found, emm87.16 and emm90.9 (GAS) and stC36.11 (SDSE). While the GAS population among children was unexpectedly clonal, substantial genetic diversity was found among the isolates recovered from young adults. Resistance to erythromycin, clindamycin and tetracycline was detected in GAS, GBS and SDSE recovered from young adults.

Conclusions. Prevalence was slightly greater among children, but persistent carriage was greater among young adults, with SDSE being the species most associated with persistence. Few sources seemed to disseminate GAS among children, since only two clonal types were found. The volunteers hosted pathogenic streptococci persistently, including macrolide-resistant strains.

Introduction. Certain nontypeable Haemophilus influenzae cannot be assigned a sequence type (ST) by Multilocus Sequence Typing (MLST) due to the lack of the fucK gene, one of seven MLST loci in H. influenzae , which encodes a fucose-operon enzyme.

Aims. To confirm whether the loss of fucK is also found in the encapsulated strains, we analysed clinical isolates of H. influenzae serotype e (Hie).

Methodology. We conducted MLST, PFGE, and antimicrobial susceptibility tests of 45 Hie strains; the majority (n=43) were derived from respiratory samples of pediatric patients at Chiba Children’s Hospital between 2000 and 2016. The two remaining strains were obtained from the blood of elderly patients with invasive H. influenzae diseases (IHiDs) between 2015 and 2016 at general hospitals. For the fucK-negative strains, PCR analysis for fucose operon was also performed.

Results. Four STs (ST18, 122, 621 and 1758) were assigned to 13 strains, and remaining 32 (including one associated with IHiD) were fucK-negative, completely missing the fucose operon. The allelic profiles of six other loci were identical among 31 strains and to that of ST18, 122 and 621, and these strains were genetically closely related. Forty of 45 isolates were ampicillin-sensitive.

Conclusions. The loss of fucK was frequently observed in clinical isolates of Hie from children. Moreover, fucK-negative Hie may be the cause of IHiD in adult patients. The majority of Hie, including fucK-negative strains, were shown to be clonally related and were ampicillin sensitive. This represents the first report examining fucK losses in encapsulated H. influenzae .

Four group A streptococcus (GAS) bacteraemia occurred in a small burn unit within 2 weeks. The GAS patient isolates, characterized as emm89, shared the same PFGE pulsotype with two other strains isolated 2 months later. The outbreak investigation revealed that a nurse was the most likely source of GAS transmission, as she was confirmed to carry the same outbreak strain in her throat and had direct and regular contact with the six outbreak patients in the unit. The outbreak was controlled after the nurse had undergone eradication treatment. This report highlights the emergence of the emm89 clone and its capacity to elicit invasive GAS outbreaks.

Introduction. Severely burned patients are susceptible to bacterial infection within their burn wounds, which frequently leads to sepsis, multiple organ failure and death. The opportunistic pathogen Pseudomonas aeruginosa , an organism inherently resistant to multiple antibiotics, is a common cause of sepsis in these patients.

Aim. Development of a topical treatment unrelated to conventional antibiotics is essential for prevention of P. aeruginosa infection and sepsis, leading to a role for the direct application of probiotics or their by-products.

Methodology. We examined the effectiveness of 20× concentrated supernatant from Lactobacillus gasseri strain 63 AM (LgCS) grown in de Man, Rogosa and Sharpe broth in inhibiting P. aeruginosa biofilms in vitro, as well as in reducing wound bioburden and P. aeruginosa sepsis in vivo.

Results. LgCS inhibited the growth of P. aeruginosa strain PAO1, prevented its biofilm development and eliminated partially developed PAO1 biofilms. In the murine model of thermal injury, a single injection of LgCS following injury and PAO1 infection reduced mortality to 0 % and prevented systemic spread (sepsis). Furthermore, a second injection of LgCS 24 h after the first eliminated PAO1 from the wound. In the murine dorsal excision infection model, either LgCS or ceftazidime treatment of the PAO1-infected wound significantly reduced the mortality rate among infected mice, while combining LgCS with ceftazidime eliminated mortality.

Conclusion. These results suggest the potential of LgCS in preventing sepsis from P. aeruginosa infection in severely burned and other immunocompromised patients.