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Distribution, virulence and antimicrobial resistance profile of Bacillus species from the environment of four public hospitals in South Africa.

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Hospital-acquired infections (HAIs) are counted as the most crucial global health crisis. The hospital environment may be colonized by opportunistic pathogens, which can lead to HAIs in hospitalized patients. As a result, microbial monitoring of the hospital environment is important in managing these pathogenic organisms. This research aimed to investigate the prevalence, antimicrobial resistance, virulence genes, and genetic diversity of Bacillus spp. on hospital surfaces in public hospitals in KwaZulu-Natal (KZN), South Africa (SA). A total of 777 swabs were collected from four different public hospitals classified as A (Central), B (Tertiary), C (Regional), and D (District), within three different wards (paediatric, general ward, and intensive care unit (ICU) and 11 touchable surfaces belonging to medical devices and well used equipment. Samples were assessed for the existence of Bacillus spp in these four public hospitals. Bacillus was isolated using the microbial plating method on a selective Bacillus medium, and the biochemical characteristics confirmed, including oxidase, catalase, motility, and triple sugar iron (TSI). Molecular confirmation was also performed using polymerase chain reaction (PCR) targeting the MotB gene for Bacillus cereus and 16s rRNA gene for Bacillus subtilis. The minimum inhibitory concentration (MIC) technique evaluated the antimicrobial resistance profile using the Clinical Laboratory Standards Institute (CLSI) guidelines. Molecular characterization was conducted for seven antimicrobial resistance genes and 11 virulence factors using PCR. Genetic relatedness between the isolates across the four hospitals was evaluated by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A total of 777 samples were collected in the hospital environments, of which 135 were positive for Bacillus spp. Species identification revealed 4 % (6) as Bacillus subtilis and 96 % (129) as B. cereus. The overall prevalence of Bacillus spp. per hospital was 24 % (32/135) for Central (hospital A), 33 % (45/135) for Tertiary (hospital B), 27 % (36/135) for Regional (hospital C) and 16 % (22/135) for District hospital (hospital D). A statistically significant difference in Bacillus prevalence (p = 0.044) was found between tertiary and regional hospitals (p = 0.000).The prevalence among the wards was averaging 33 % (45/135), noting ICU with the highest prevalence of 35 % (47/135). In terms of the wards, no statistically significant differences were found (p = 0.133). The hospital and the wards had a strong correlation (r = 0.525, p = 0.000).The highest prevalence on frequently touched sites was a bed with 15 % (20/135), drip stands and sinks with 12 % (16/135) respectively, ward phones with 11 % (15/135), and nurses’ tables with 10 % (14/135). Complete resistance was observed against ampicillin (100 %; 135/135), and high resistance against ciprofloxacin (99 %; 134/135), amoxicillin (97 %; 131/135), tetracycline (82 %; 112/135), cefotaxime (51 %; 69/135), and erythromycin (43 %; 59/135). Lower resistance was observed against meropenem (20 %; 27/135) and imipenem (25 %; 26/135). A total of 43 different antibiograms were detected, with 97 % (132/135) of the isolates found to be multidrug resistance (MDR). No statistically significant difference was observed between the antibiotics tested (p ≥ 0.05). The observed resistance genes were ermB (56 %; 33/59) (conferring erythromycin resistance), tetracycline resistance-conferring genes were tetM (5 %; 5/112) and tetK (4 %; 4/112). No tetA, tetB, tet39, and the blm gene (beta-lactamase resistance-conferring gene) were detected. Different toxins produced by Bacillus are associated with the pathogenicity of Bacillus species. Prevalence of the virulence enterotoxin genes associated with diarrhea prevailed in 88 % (99/135) of hblD, 77 % (104/135) in hblA and CytK respectively, nheC 67 % (90/135), nheB 65 % (88/135), nheA 64 % (89/135), hblC 55 % (74/135), bceT 44 % (59/135), hlyII 37 % (50/135), and finally EntFM 27 % (37/135) of the isolates housed the gene. No cereulide (ces gene) causing emetic syndrome was detected. Typing using ERIC-PCR noted type clusters composed of isolates from different wards, environmental sites, and equipment and showing high genetic diversity, indicating no common infection sources. This study revealed a moderate prevalence of Bacillus spp. collected from the environment of the four public hospitals. High resistance was observed for some antibiotics that are usually effective against Bacillus; this may serve as a potential risk for effective treatment of Bacillus infections. Most isolates harboured virulence factors that cause diarrheal syndrome rather than those causing the emetic syndrome. These findings highlight the need for microbial surveillance of hospital environments to inform and improve current intervention programs for cleaning methods in public hospitals to reduce environmental contamination and transmission of pathogenic Bacillus spp. infections associated with HAI’s in hospitalised patients.


Masters Degree. University of KwaZulu-Natal, Durban.