The clinical impact of antimicrobial resistance genomics in competition with she-camels recurrent mastitis metabolomics due to heterogeneous Bacilluslicheniformis field isolates

Abstract

Background and aim: Recently, cases of mastitis refractory to treatment have been reported frequently. There are limited routine laboratory investigations on Camelidae infections. Mastitis has been estimated to affect more than 25% of lactating she-camel with up to 70% milk loss. The details of Bacillus spp. pathogenesis in mastitis are not yet fully described. The present study is the first detailed phenotypic and genotypic characterization of Bacillus licheniformis isolates from recurrent mastitic she-camels with sepsis in Egypt.

Materials and methods: The udders of 100 she-camels were investigated, samples collected from smallholders' farmers in 10 localities within three governorates in Egypt: Marsa Matrouh, Giza, and Sharkia governorates. The pathogens ascend from udder inducing abortion at different trimesters of pregnancy. Polymerase chain reactions-mediated proofs of identity were applied for diagnostic and taxonomic purposes, where the 16S rRNA gene sequence and the β subunit of RNA polymerase encoding gene rpoB are the molecular targets.

Results: The genetic elements classified the subspecies to B. licheniformis 61.4%, in addition to, Corynebacterium bovis 29.8%. The somatic cell count (≤1×10⁷ cells/ml) and California mastitis test reactivity (+3 or +4) of milk clinically classified the she-camels population (n=100) under investigation into 50, 20, and 30 as healthy, subclinical, and clinical mastitic she-camels, respectively. During bacterial isolation, 80 species were noticed, of which 71.25% (57/80) and 28.75% (23/80) were Gram-positive and negative, respectively, in two clinical forms: Single (40%, n=16/40) and mixed (60%, n=34/40) bacterial infections. In vitro, 100% sensitivity for gentamycin (10 µg) and ofloxacin (5 µg) was noted; however, it was reduced to 50%. Moreover, during in vivo treatments, cloxacillin (5 µg) upraised as the most effective alternative with 90% sensitivity.

Conclusion: Neither recurrent mastitis nor Bacillus species are thoroughly investigated with regard to reproduction performance in Egypt and the usefulness of these strains as antimastitis probiotics. Both persistent bacteremia and dormant endospores were formed but unaffected by standard schemes of antimicrobials injections which proposed the risk of pathogenic bacilli contaminating row milk from apparently healthy she-camel. The discrepancies between treatment results were induced by the resistance that started to develop by the organisms due to frequent and/or faulty use of applied antibiotics.

Keywords: 16S rRNA gene; Bacillus species; Camelidae; Egypt; antimicrobial resistance; probiotics; recurrent mastitis; rpoB gene.

Figure-1

The in vitro antibiotic reactivity’s of the two predominant Gram-positive bacilli-purified colonies of Bacillus licheniformis and Corynebacterium bovis from lactating she-camels milk samples.

Figure-2

The recovery profile of the lactating she-camels in response to treatment with antibiotics against predominant bacterial isolates from milk microbiota of subclinical and/or clinical cases.

Figure-3

Molecular identified Bacillus licheniformis and Corynebacterium bovis pure colonies isolated from recurrent mastitic she-camels udders represented by polymerase chain reaction (PCR) products; (a and b) lane 1 and 2: 1403 bp bands, and lanes 8 and 9: 850 bp bands are amplifications of 16S rRNA genes. Lanes 3, 4, 5, and 10: Are blank controls negative. Lanes 2 and 7: Are PCR products of controls positive Corynebacterium pyogenes and Bacillus subtilis strains. Lanes M: are Gene Ruler 100 bp Plus DNA ladder.