Subspecies Typing of Streptococcus agalactiae Based on Ribosomal Subunit Protein Mass Variation by MALDI-TOF MS.

Background: A ribosomal subunit protein (rsp)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was developed for fast subspecies-level typing of Streptococcus agalactiae (Group B Streptococcus, GBS), a major cause of neonatal sepsis and meningitis. Methods: A total of 796 GBS whole genome sequences, covering the genetic diversity of the global GBS population, were used to in silico predict molecular mass variability of 28 rsp and to identify unique rsp mass combinations, termed "rsp-profiles". The in silico established GBS typing scheme was validated by MALDI-TOF MS analysis of GBS isolates at two independent research sites in Europe and South East Asia. Results: We identified in silico 62 rsp-profiles, with the majority (>80%) of the 796 GBS isolates displaying one of the six rsp-profiles 1-6. These dominant rsp-profiles classify GBS strains in high concordance with the core-genome based phylogenetic clustering. Validation of our approach by in-house MALDI-TOF MS analysis of 248 GBS isolates and external analysis of 8 GBS isolates showed that across different laboratories and MALDI-TOF MS platforms, the 28 rsp were detected reliably in the mass spectra, allowing assignment of clinical isolates to rsp-profiles at high sensitivity (99%) and specificity (97%). Our approach distinguishes the major phylogenetic GBS genotypes, identifies hyper-virulent strains, predicts the probable capsular serotype and surface protein variants and distinguishes between GBS genotypes of human and animal origin. Conclusion: We combine the information depth of whole genome sequences with the highly cost efficient, rapid and robust MALDI-TOF MS approach facilitating high-throughput, inter-laboratory, large-scale GBS epidemiological and clinical studies based on pre-defined rsp-profiles.

Maternal vaginal colonization with selected potential pathogens of neonatal sepsis in the era of antimicrobial resistance, a single center experience from Sri Lanka.

BACKGROUND: Maternal vaginal colonization with antibiotic resistant organisms is a growing concern in countries with high antibiotic resistance rates. METHODS: A low vaginal swab was collected from mothers on admission, on discharge and a peri-rectal swab was collected from the neonates born to these mothers on discharge. Routine microbiological methods were used to identify the colonization rates for Escherichia coli, Klebsiella spp. and Streptococcus agalactiae. RESULTS: The pre-delivery colonization rate among the 250 participants for total Enterobacteriaceae was 18.8%. The colonization rates for Klebsiella spp., E. coli and S. agalactiae were, 12.4, 5.6 and 14.8% respectively. Two Klebsiella spp. and two E. coli isolates were confirmed to be exentend spectrum ß lactamase (ESBL) producers with the commonest resistant determinant being blaCTX-M. Post-delivery swabs were collected from 130 participants and the colonization rates were 41.5% for Enterobacteriaceae, 25.4% for Klebsiella spp., 10.8% for E. coli, and 10.8% for S. agalacteiae. Three Klebsiella isolates and one E. coli isolate were confirmed to be ESBL producers with the commonest resistant determinant being blaCTX-M. Considering the 130 participants with both samples, there was a significant increase in the colonization with any Enterobacteriaceae and Klebsiella spp. (p < 0.05). Peri-rectal swabs were collected from neonates in 159 instances. The isolation rates for Enterobacteriaceae was 34%. The genus specific isolation rate for Klebsiella was 21.4% while the rates for E. coli and S.agalactiae were 10.1 and 5.7% respectively. Two of the E. coli were confirmed to be ESBL producers while none of the klebsiellae were identified to be so. Considering these 159 instances where both the mother and baby were sampled, random amplification of polymorphic DNA (RAPD) analysis revealed that Enterbacteriaceae with same strain type was present in 6.9% of the instances, indicating possible transfer between the mother and neonate. The transfer rate for ESBL producers were 0.6%. CONCLUSIONS: The lower level of antimicrobial resistance among these potentially community acquired isolates is encouraging. However, in view of the increasing level of resistance reported elsewhere in the region, regular monitoring is warranted.

Spread of resistant gram negatives in a Sri Lankan intensive care unit.

BACKGROUND: Infections with multi drug resistant (MDR) organisms are a major problem in intensive care units (ICUs). Proper infection control procedures are mandatory to combat the spread of resistant organisms within ICUs. Well stablished surveillance programmes will enhance the adherence of the staff to infection control protocols. The study was conducted to assess the feasibility of using basic molecular typing methods and routine hospital data for laboratory surveillance of resistance organisms in resource limited settings. METHODS: A retrospective study was conducted using consecutive Gram negative isolates obtained from an ICU over a six month period. Antibiotic sensitivity patterns and random amplified polymorphic DNA (RAPD) based typing was performed on the given isolates. RESULTS: Of the seventy isolates included in the study, seven were E.coli. All E.coli were MDRs and Extended Spectrum ß lactamse (ESBL) producers carrying bla CTX-M. Fourteen isolates were K.pneumoniae, and all were MDRs and ESBL producers. All K.pneumoniae harboured bla SHV while 13 harboured bla CTX-M. The MDR rate among P.aeruginosa was 13% (n=15) while all acinetobacters (n=30) were MDRs. Predominant clusters were identified within all four types of Gram negatives using RAPD and the ICU stay of patients overlapped temporally. CONCLUSION: We propose that simple surveillance methods like RAPD based typing and basic hospital data can be used to convince hospital staff to adhere to infection control protocols more effectively, in low and middle income countries.