Molecular microbial ecology of hospital ward environments.

Evidence indicates that the hospital environment plays a role in hospital acquired infections (HAI). Previous studies have focused on outbreaks and the role of specific culturable pathogens. There is still considerable debate about the role of the environment and the importance of cleaning, therefore more comprehensive culture-independent microbial ecology studies are required.This study aimed to utilise culture-independent methods to characterise the microbial communities in an orthopaedic ward and theatre environment under normal operating conditions, to determine the distribution of different microorganisms, their viability and persistence following routine cleaning. Culture was used to quantify culturable bacteria present and select bacterial populations of interest. Antibiotic-resistance gene specific PCRs were used to investigate the presence of resistance determinants. PCR-DGGE was used to resolve fragments of the 16S rRNA gene from total DNA extractions and the resulting sequence data was used to identify microorganisms present. Cell viability was assessed using RNA as the template for reverse transcriptase PCR-DGGE. Quantification of bacteria on environmental surfaces using culture indicated that near patient surfaces rarely exceeded the recommended 2.5 CFU/cm2 limit, providing evidence that current cleaning regimes employed on these wards are sufficient to keep microbial contamination low. PCR-DGGE showed that sequences similar to Staphylococci dominated the environment, particularly S. hominis, and S. haemolyticus, which were retrieved from the floor before and immediately after cleaning. Antibiotic gene specific PCRs were used to demonstrate the presence of the mecA gene, aminoglycoside modifying enzyme genes and qac genes in isolated environmental non-aureus Staphylococci. This data has highlighted the potential role of this environment as a reservoir of pathogenic Staphylococci.Using PCR-DGGE the orthopaedic wards yielded sequences from DNA and RNA templates similar to species associated with human skin and large intestine. While Staphylococci were shown to dominate most environmental sites, Kocuria and Corynebacterium species were found to be specifically associated with the bed rails. Few Gram negative species were detected by molecular methods or selective culture in the ward environment. Sequences similar to Cupriavidus and Chryseobacterium species were detected in the operating theatre environment using PCR-DGGE, in contrast using culture; Staphylococci were readily isolated from the theatre environment. PCR-DGGE was also used to investigate the presence of fungi in sink drains on an orthopaedic and intensive care ward. While Fusarium species were widely distributed on both wards, sequences similar to Candida were only detected in samples from the ICU wards using PCR-DGGE. When culture was used Candida species were readily grown from both environments. The differences between the species detected using culture and PCR-DGGE suggest that the two techniques should be used together to provide complementary data. Overall PCR-DGGE has been used for direct identification of the dominant viable bacteria and fungi in the hospital environment, to provide an estimate the relative abundance of species, and give an overview of the effect of routine ward cleaning on the bacterial community