Colistin susceptibility of gram-negative clinical isolates from Tamil Nadu, India

All 94 clinical isolates included in the present study were from two different regions in Tamil Nadu; Chennai and Trichy, and were collected between March and July 2014. All the isolates identified to be multidrug resistant using a disk diffusion method in clinical centers were received in vials from both Chennai (Hitech Diagnostic Centre) and Trichy (Doctor’s Diagnostic Centre). Anonymized isolates (unlinked to patient data) were received at the Antimicrobial Resistant Laboratory, School of Bio Sciences and Technology in VIT University, Vellore, India. All isolates were subcultured onto brain-heart infusion agar (HiMedia Laboratories, Mumbai, India) and stored at –80°C for further analysis.

Minimal inhibitory concentrations (MICs) for colistin and meropenem were determined using a microbroth dilution method. Briefly, Mueller Hinton (MH) broth No. 2 with controlled cations (HiMedia Laboratories) was prepared and 100 μl was aliquoted into each of the 96 wells in a microtiter plate. A total of 100 μl of broth with antibiotic with concentrations ranging from 0.25 μg/ml to 256 μg/ml were made and a single well in each row was allocated as a growth control without antibiotic. Then 100 μl of MH broth with bacteria (to have final count of 5 × 10⁴ ml) prepared from an overnight culture of a single colony inoculated into MH broth No. 2 with controlled cations was added. Microtiter plates were incubated at 37°C for 18 h. An MIC value ≥ 8 μg/mL was considered to be the resistance breakpoint.

DNA was extracted from all the isolates using a boiling method. Briefly, 500 μL bacteria grown overnight were centrifuged at 10,000 rpm for 5 min, and to the pellet 100 μL of sterile distilled water was added and heated for 10 min at 95°C. The supernatant was then used as a template. The 16S rRNA gene from each isolate was amplified and the amplicons were sent to Macrogen, South Korea for sequencing. Sequence homologies were determined using a nucleotide BLAST analysis at www.ncbi.nlm.nih.gov/ BLAST and identified at a species level.

Identification of all 94 isolates included in the present study was determined by 16S rRNA gene sequencing. The results showed that among 94 isolates, 48 were E. coli, 9 K. pneumoniae, 10 P. aeruginosa, 5 Proteus mirabilis, 4 Salmonella enterica, 3 Enterobacter hormaechei, 3 Enterobacter cloacae, 2 Achromobacter xylosoxidans, 2 Acinetobacter baumannii, 1 Providencia vermicola, 1 Acinetobacter towneri, 1 Enterobacter gergoviae, 2 Providencia rettgeri, 1 Enterobacter asburiae, 1 Pseudomonas stutzeri, and 1 Salmonella typhi.

All the isolates sent to our laboratory were multidrug resistant as determined by disk diffusion. All multidrug resistant isolates tested against colistin with concentrations ranging from 0.12 μg/mL to 128 μg/mL showed MIC₅₀ 1 μg/mL and MIC₉₀ >128 μg/mL, respectively. Colistin resistant isolates (Figure 1) included E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, P. mirabilis, E. cloacae, P. rettgeri, S. enterica.

Figure 1

Among 27 colistin resistant isolates 14 (52%) were resistant also to meropenem. A total of 15% isolates were found to be resistant to both meropenem and colistin. Isolates with dual resistance were E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. For colistin MIC₅₀ was 1 μg/mL and MIC₉₀ was >128 μg/mL, for meropenem MIC₅₀ < 0.06 μg/mL and MIC₉₀ > 128 μg/mL (Table 1).