Enterococcus sp. contamination surveillance in different levels of healthcare in eThekwini District, KwaZulu-Natal (KZN) South Africa.

Abstract

Hospital-acquired infections (HAIs) have been identified as long-standing setbacks affecting hospitals' quality of health care. While one of the major challenges related to HAIs is controlling cross-transmission, the role and significance of the inanimate hospital environment chain of transmission are yet to be unequivocally elucidated. Therefore, this study investigated the functional profile and diverseness of bacteria from various inanimate environmental sources, from two different wards in public hospitals at various healthcare levels in eThekwini District, KwaZulu-Natal, South Africa. True to the study focus on investigating the dissemination of bacteria from equipment within the hospital, the study further used Enterococcus as well-known HAI as target bacteria and described the molecular and genomic profiles of this specie isolated from the hospital environments. Samples were collected for a period of three months (September – November 2017) from the four levels of healthcare in eThekwini district, KwaZulu-Natal. The intensive care unit and peadiatic ward were employed in this study. An overall of 620 swabs were collected from areas frequently touched by healthcare workers (HCWs) and patients. These sites include the occupied bed linen, unoccupied bed linen, drip stands, patient files, ward phones, ventilators, nurses' tables, blood pressure apparatus, sinks, linen room door handle and mops. Swabs were placed in Amies transport medium and transported in a cooler box to the laboratory facility to be processed within four hours. The collected swabs (n=620) were pooled and incubated in tryptone soya broth containing 6.5% NaCl at 36.5oC for 24 hrs and subsequently plated on enterococci chromogenic media. The microbial diversity and functional profiles from the sites were identified using 16S rRNA metagenomics. Positive colonies were sub-cultured on bile esculin azide agar, and screened using standard microbiological methods, including haemolytic, oxidase and catalase, and API. Identifications were confirmed with polymerase chain reaction (PCR) with the added genus-specific tuf-gene and species-specific sodA-gene. Antibiotic resistance patterns in the Enterococcus spp. isolates were determined by the Kirby-Bauer disk diffusion method against 14 antibiotics as recommended by the Clinical and Laboratory Standard Institute (CLSI) guidelines.

Thirty-seven samples from E. faecalis showed intermediate Resistance to vancomycin and were further analyzed using molecular tools viz. whole-genome sequencing (WGS) and bioinformatics analyses. This enabled determining the resistome, mobile genetic elements (MGEs), and clonal lineages circulating across the sites, wards, and hospitals. Metagenomics identified a total of 288 species, 190 genera, 105 families, 50 orders, 29 classes and 11 phyla from the samples analyzed. The dominant functional metabolic pathways implicated in causing human infection discovered were the signal transduction mechanisms, citrate cycle (TCA), transcription-factor bisphenol degradation, tyrosine metabolism. A total of 295 Enterococcus spp. isolates were recovered from the hospitals` environmental sites, 83% (n=245) were identified as Enterococcus faecium, 13% (n=38) as Enterococcus faecalis, 2% (n=6) Enterococcus gallinarum and another 2% (n=6) Enterococcus casseliflavus. Notably, the pediatric wards had the highest isolation rate compared to ICU, 64% and 36%, respectively. Overall, the sites with the highest isolation rate were occupied beds and mops (to clean ward floors) with 14.9% (n=44) each. The tertiary hospital were the most affected. The most prominent MDR antibiogram for E. faecium was CIP-RIF-NIT-TET-ERY and for WGS analysis of the E. faecalis samples confirmed that the tet(M) and erm(C) genes were the prevalent antibiotic resistance genes found in hospitals. The isolates harboured mobile genetic elements consisting of plasmids (n =11) and prophages (n=14), predominantly clonally specific. The 37 isolates analyzed consisted of 15 clonal lineages with six major sequence types (ST). Phylogenomic analysis showed that major lineages were mostly conserved within specific hospital environments. This study highlighted the inanimate hospital environment as a possible source of opportunistic nosocomial pathogens using Enterococcus as an illustrative example and emphasized the urgent necessity to optimize infection prevention and control measures to intercept/moderate the spread of bacteria in the hospital environments.