- Volume 64, Issue 6, 2015

The current Ebolavirus disease (EVD) epidemic in West Africa has now been running for >1 year and has been an international health emergency for >6 months. As the weekly number of new cases falls, the World Health Organization is preparing its response to the final stages of the epidemic. The final totals will exceed 20 000 cases and 8000 deaths. An ability to adapt disease countermeasures including laboratory support to the changing epidemiology of EVD has become a matter of urgency. This article considers the planning, development and modification of a flexible microbiology laboratory response, and describes logistic and operational considerations for clinical and public health microbiologists.

The ica operon encoding polysaccharide intercellular adhesion, which facilitates biofilm formation in staphylococci, has been extensively studied in Staphylococcus epidermidis and Staphylococcus aureus. Based on in silico analysis, we suggest the following functional model for Ica proteins in S. capitis. IcaA is responsible for polysaccharide synthesis. IcaA and IcaD complete transferring the growing sugar chain to the cell surface; IcaB is a deacetylase, with the same function as IcaB of S. epidermidis. IcaC mainly modifies the synthesized glucan by acetylation. We also examined the effects of subinhibitory concentrations of erythromycin on phenotypic biofilm expression and transcription of biofilm-related genes, using isolates representing the two subspecies of Staphylococcus capitis and different biofilm and resistance phenotypes. On induction with erythromycin, biofilm density was strongly elevated in two erythromycin-resistant S. capitis, but not in three susceptible isolates. In the representative erythromycin-resistant S. capitis subsp. urealyticus, there were significant upregulations of the icaA gene and its positive regulator sarA on transition to the stationary phase without erythromycin induction. There were also significant increases in the transcription levels of icaA, rsbU and sigB corresponding to a very strong biofilm phenotype in the stationary phase on erythromycin stress. In contrast, the representative erythromycin-susceptible S. capitis subsp. capitis displayed upregulation only of altE on entry into the stationary phase with erythromycin induction, but this change was not associated with enhancement of biofilm production. These findings suggest that the two subspecies of S. capitis adopt different pathogenesis and survival strategies to adapt to a hostile environment.

Urinalysis culminates in a workload skew within the clinical microbiology laboratory. Routine processing involves screening via manual microscopy or biochemical dipstick measurement, followed by culture for each sample. Despite this, as many as 80 % of specimens are reported as negative; thus, there is vast wastage of resources and time, as well as delayed turnaround time of results as numerous negative cultures fulfil their required incubation time. Automation provides the potential for streamlining sample screening by efficiently (>30 % sample exclusion) and reliably [negative predictive value (NPV) ≥ 95 %] ruling out those likely to be negative, whilst also reducing resource usage and hands-on time. The present study explored this idea by using the sediMAX automated microscopy urinalysis platform. We prospectively collected and processed 1411 non-selected samples directly after routine laboratory processing. The results from this study showed multiple optimum cut-off values for microscopy. However, although optimum cut-off values permitted rule-out of 40.1 % of specimens, an associated 87.5 % NPV was lower than the acceptable limit of 95 %. Sensitivity and specificity of leukocytes and bacteria in determining urinary tract infection was assessed by receiver operator characteristic curves with area under the curve values found to be 0.697 [95 % confidence interval (CI): 0.665–0.729] and 0.587 (95 % CI: 0.551–0.623), respectively. We suggested that the sediMAX was not suitable for use as a rule-out screen prior to culture and further validation work must be carried out before routine use of the analyser.

Candida glabrata is currently ranked as the second most frequently isolated aetiological agent of human fungal infections, next only to Candida albicans. In comparison with C. albicans, C. glabrata shows lower susceptibility to azoles, the most common agents used in treatment of fungal infections. Interestingly, the mechanisms of resistance to azole agents in C. albicans have been much better investigated than those in C. glabrata. The aim of the presented study was to determine the mechanisms of resistance to azoles in 81 C. glabrata clinical isolates from three different hospitals in Poland. The investigation was carried out with a Sensititre Yeast One test and revealed that 18 strains were resistant to fluconazole, and 15 were cross-resistant to all other azoles tested (voriconazole, posaconazole and itraconazole). One isolate resistant to fluconazole was cross-resistant to voriconazole, and resistance to voriconazole only was observed in six other isolates. All strains were found to be susceptible to echinocandins and amphotericin B, and five were classified as resistant to 5-fluorocytosine. The sequence of the ERG11 gene encoding lanosterol 14-α demethylase (the molecular target of azoles) of 41 isolates, including all strains resistant to fluconazole and three resistant only to voriconazole, was determined, and no amino acid substitutions were found. Real-time PCR studies revealed that 13 of 15 azole-resistant strains showed upregulation of the CDR1 gene encoding the efflux pump. No upregulation of expression of the CDR2 or ERG11 gene was observed.

The increasing emergence of multidrug-resistant Acinetobacter baumannii constitutes a worldwide threat in hospital settings. Efflux-mediated resistance, particularly the resistance-nodulation-division (RND)-type efflux pumps, contributes significantly to decreased susceptibility to multiple antibiotics when overexpressed. Using PCR-based detection, the prevalence of genes encoding the RND efflux pumps AdeB, AdeJ and AdeG was investigated amongst 144 epidemiologically characterized and geographically diverse A. baumannii isolates of worldwide origin, representing International Clones 1–8 and genotypically unique isolates. Furthermore, five putative RND-type efflux genes identified via an in silico approach were included. Five of the eight investigated efflux pump genes were present in all isolates, including adeJ and adeG; the prevalence of the others varied between 65 and 97 %. No association between the presence of one or multiple pumps to a specific clonal lineage was detected. The high prevalence of the efflux pump genes supports a fixed function of each individual pump that is not yet fully understood.

A healthy vaginal environment is predominated by certain Lactobacillus species, which lead to the prevention of infections of the reproductive tract. This study examined the characteristics of cultivable Lactobacillus species in both healthy women and women with bacterial vaginosis (BV). Between November 2011 and September 2013, 139 women attending a women's clinic in Mysore, India, were evaluated for BV in a cross-sectional study. BV was diagnosed using Amsel's criteria: homogeneous vaginal discharge, vaginal pH >4.5, production of amines, and presence of “clue” cells. Those with three or more of the characteristics were considered to have BV. Vaginal swabs were then cultured in Rogosa agar and de Man-Rogosa-Sharpe broth. Gram-positive lactobacilli generating 600–800 bp amplicons by16 sRNA were further characterized by sequencing. Cultivable vaginal samples were obtained from 132 women (94.9 %). According to the Amsel criteria, 83 women (62.1 %) were healthy, and 49 (37.1 %) had BV. Eleven different Lactobacillus species were isolated from 47 women. The common lactobacilli species found in this sample included L. crispatus (39.6 %), L. gasseri (45.8 %), and L. jensenii (14.6 %). Lactobacilli were isolated from 39 healthy women and eight with BV. L. gasseri was cultured from 18.8 % of healthy women and 6.1 % with BV. The presence of L. reuteri was significantly associated with normal vaginal microbiota (P-value = 0.026). These results further our understanding of vaginal lactobacilli colonization and richness in this particular population. Our findings showed that lactobacilli species present in the vaginas of healthy women in India do not differ from those reported from other countries.

Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases.