This study was carried out to elucidate the role of reduced antibiotic penetration in the resistance of Staphylococcus aureus and Staphylococcus epidermidis biofilms to different antibiotics. The biofilms of S. aureus ATCC 29213 and S. epidermidis ATCC 35984 were grown on black, polycarbonate membranes (diameter, 13 mm; pore size, 0.4 µm) placed on tryptic soy agar plates at 37°C for 48 h. The penetration of oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin through the biofilms was determined by measuring the diameter of zones of growth inhibition (of S. aureus ATCC 25923, a quality control strain) on Mueller–Hinton agar plates following diffusion of each antibiotic from an overlying antibiotic disc through the biofilm to the agar medium versus the respective control assemblies. The penetration of oxacillin and cefotaxime (β-lactams) and vancomycin (a glycopeptide) was significantly reduced through S. aureus and S. epidermidis biofilms whereas that of amikacin (an aminoglycoside) and ciprofloxacin (a fluoroquinolone) was unaffected. The results of this study indicate that the role of reduced antibiotic penetration in the drug resistance of S. aureus and S. epidermidis biofilms may vary with the antibiotic being used.

The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms. This study investigated whether persisters and/or SCVs contribute to the antibiotic resistance ofStaphylococcus aureusbiofilms. A detailed dose-dependent killing of biofilms and planktonic cells with five antibiotics (oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin) was analysed by treating them with each antibiotic at a concentration of 0–100 μg ml⁻¹at 37 °C for 48 h. The killing of biofilm cells by all of the antibiotics showed the presence of persister cells – most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re-exposure to the antibiotics. SCVs were observed in biofilms only after treatment with ciprofloxacin, and these SCVs were of a transient nature. The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. The treatment of planktonic cells with amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. Thus the high resistance ofS. aureusbiofilms to multiple unrelated antibiotics is largely dependent on the presence of persister cells. Biofilms harbour a large number of persisters in comparison with planktonic cultures, which either do not harbour persisters or harbour only a small number. SCVs, although not specifically associated withS. aureusbiofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associated infections. The intrinsic resistance of these variants may in turn contribute to the enhanced antibiotic resistance of the biofilms thus formed.

The role ofStaphylococcus aureussmall-colony variants (SCVs) in the pathogenesis of biofilm-associated infections remains unclear. This study investigated the mechanism behind increased biofilm-forming potential of a menadione-auxotrophicStaphylococcus aureusSCV compared with the wild-type parental strain, as recently reported by our laboratory. SCVs displayed an autoaggregative phenotype, with a greater amount of polysaccharide intercellular adhesin (PIA), significantly reduced tricarboxylic acid cycle activity and a decreased susceptibility to aminoglycosides and cell-wall inhibitors compared with wild-type. The biofilms formed by the SCV were highly structured, consisting of large microcolonies separated by channels, and contained more biomass as well as significantly more PIA than wild-type biofilms. The surface hydrophobicity of the two phenotypes was similar. Thus, the autoaggregation and increased biofilm-forming capacity of menadione-auxotrophicStaphylococcus aureusSCVs in this study was related to the enhanced production of PIA in these variants.

Background & objectives: The increasing prevalence of extended-spectrum β-lactamases (ESBLs) has abated therapeutic options worldwide. This study was undertaken to investigate the molecular profile and resistance patterns of ESBLs among clinical isolates of Escherichia coli and Klebsiella pneumoniae at four tertiary care centres in India. Methods: Clinical isolates of E. coli and K. pneumoniae were collected from the All India Institute of Medical Sciences (AIIMS), New Delhi; the Jawaharlal Institute of Postgraduate Medical Education & Research (JIPMER), Puducherry; Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh and Christian Medical College (CMC), Vellore, over one and a half year period. Antimicrobial susceptibility was determined by Kirby-Bauer disc diffusion method. ESBLs were confirmed phenotypically, and multiplex PCR was performed to identify genes for β-lactamases (bla _TEM, bla _SHV, bla _OXA-1, bla _CTXM-1, bla _CTXM-2, bla _CTXM-9 and bla _CTXM-15). Results: Among 341 E. coli isolates collected during the study period, 171 (50%) harboured bla _TEM, 145 (43%) bla _OXA-1, 70 (21%) bla _CTXM-1, 19 (6%) bla _SHV and four (1%) harboured bla _CTXM-2. Phenotypically, combined disc test detected ESBL production in 98/298 (33%) E. coli. Among 304 K. pneumoniae isolates, 115 (38%), 89 (29%), 83 (27%), 64 (21%) and two (0.6%) harboured bla _TEM, bla _OXA-1, bla _CTXM-1, bla _SHV and bla _CTXM-2, respectively. Combined disc test (CDT) detected ESBL production in 42 per cent K. pneumoniae. Most of the bla _CTXM-1 positive isolates were also bla _CTXM-15 positive. The carbapenem susceptibility ranged from 56 to 88 per cent for E. coli and from 20 to 61 per cent for K. pneumoniae. Antibiotic sensitivity patterns showed that colistin (CST) was the most sensitive drug for both E. coli (271/274, 99%) and K. pneumoniae (229/234, 98%). Interpretation & conclusions: The prevalence of ESBL among four study centres varied, and bla _TEM, bla _OXA-1 and bla _CTXM-15 were the most common genotypes in E. coli and K. pneumoniae isolates in India. The growing carbapenem resistance and emerging colistin resistance warrant the judicious use of these antimicrobials.

Candidaemia is a major cause of morbidity and mortality of hospitalised patients, especially in developing countries. This study was conducted to monitor any change in species distribution and antifungal susceptibility pattern of Candida species causing candidaemia over the last 20 years. The candidaemia cases reported during January 1999 and December 2018 at our centre were reviewed. The yeasts were identified by phenotypic characters (during 1999-2014) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) (during 2014-2018). Antifungal susceptibility testing (AFST) was performed in accordance with the Clinical and Laboratory Standards Institute guidelines. A total of 602,963 blood samples from patients with suspected sepsis were processed. Candidaemia was diagnosed in 7927 (1.31%) cases. The frequency of cases rose significantly (P = 0.000) in the last quarter of the study. Candida tropicalis (40.1%) was the most common species, followed by Candida albicans (15.2%), Wickerhamomyces anomalus (13.1%), Candida krusei (6.6%), Candida parapsilosis (4.7%) and others. Rare species such as Candida auris, Candida lambica, Candida orthopsilosis, Candida vishwanathii were identified after the introduction of MALDI-TOF. The minimum inhibitory concentrations of amphotericin B rose significantly from the first to last quarter (0.5%-4.9%). Fluconazole resistance was fairly constant at 7.4%-8.8%. Local epidemiology of candidaemia at our centre was distinct regarding prevalence and change of spectrum of species. The identification of rare species was possible after the introduction of MALDI-TOF. With the emergence of multidrug-resistant C. auris and resistance in other species, routine AFST has become imperative.