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Multivariate Effects of SNPs on Environmental Streptococcal Mastitis Evaluated With an NGS-Based Association Study Using Targeted Resequencing in the Bovine MHC Region.

Mastitis is an inflammatory reaction caused by bacterial infection of the teat, and a relationship between its onset and cattle major histocompatibility complex (BoLA) region has been reported. However, no comprehensive genetic analysis of mastitis caused by environmental streptococci has been reported. Here, we resequenced the BoLA region using a hybridisation capture target next-generation sequencing (NGS) method to identify disease susceptibility markers mapped to the BoLA region in environmental streptococcal mastitis. This study examined 75 cows with mastitis caused by environmental streptococci selected from 1641 cows with mastitis and 222 healthy cows without mastitis in Japan. Targeted sequences obtained from MiSeq NGS were aligned to the bovine reference genome (ARS-UCD1.2/bosTau9), and 2,920,355 variants were detected within the BoLA region of the 297 Holstein cattle. In an association study using 2264 variants after quality control, the top 20 variants with the lowest P values were selected and assigned to the 18 surrounding candidate genes, and a gene network analysis of these genes resulted in the narrowing down of five candidate genes POU5F1, IER3, GNL1, ABCF1, and PRR3. Multivariate effect analysis of all 6 SNPs associated with these 5 genes revealed that they were significantly correlated with mastitis, indicating that they were useful for classification of mastitis-resistant and mastitis-susceptible cattle. This is the first report to identify SNPs associated with environmental streptococcal mastitis with an NGS-based association study using targeted resequencing in the BoLA region, and understanding host factors may provide important clues for mastitis control.

Animals

A Pilot Study: Developing a Lactating Dairy Goat Model to Study Staphylococcus aureus Mastitis in Women.

INTRODUCTION: Lactational mastitis is common in lactating women, with Staphylococcus aureus as the most commonly isolated agent associated with infectious lactational mastitis. Currently, there are no evidence-based guidelines for antimicrobial treatment due to barriers in obtaining pharmacokinetic data from lactating women. To overcome this barrier, a suitable large animal model is needed. Goats are an ideal translational model for human mastitis due to their anatomical and physiological similarity to humans. The objective of this pilot study was to assess if goats would develop clinical mastitis following intramammary inoculation with a clinical human isolate of S. aureus with the goal of establishing an alternative in vivo model for future research. The hypothesis was that the infected mammary gland half would show similar clinical signs to women with mastitis and demonstrate a similar local immune response when compared to the control mammary gland half. METHODS: One half of the mammary gland of two healthy lactating does was inoculated with a clinical human isolate of S. aureus. The other half of the mammary gland was sham inoculated with sterile buffered saline. Physical examinations, mammary gland assessments, and sterile milk samples were collected every 12 hours post inoculation. At 96 hours post inoculation, the goats were euthanized, and the mammary glands were examined for pathological changes. RESULTS: Goats did not develop systemic signs of disease following inoculation. Focal infected mammary gland changes included warmth, swelling, redness, discoloration, and reduced milk production; the other mammary gland half remained normal throughout the study period. S. aureus was enumerated from only the infected mammary gland half. The microscopic findings of the infected half showed neutrophilic inflammation and cell necrosis consistent with acute mastitis. DISCUSSION: This pilot study demonstrated lactating does can develop clinical signs like those observed in women. Goats have the potential to be a promising animal model to study infectious lactational mastitis.

Animals

Long non-coding RNAs link DNA methylation to immune regulatory networks in bovine subclinical mastitis.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of inflammatory and immune signaling, yet their contribution to bovine subclinical mastitis remains poorly defined. Here, we characterized the lncRNA expression landscape associated with disease in milk somatic cells of healthy and subclinical mastitic Vrindavani cattle. We identified 11,403 high-confidence lncRNAs, of which 104 were differentially expressed in subclinical mastitis (adjusted P&#x2009;<&#x2009;0.05; |log2FC| &#x2265; 1), with the vast majority upregulated in mastitic samples. Predicted cis- and trans-associated target analyses identified 637 non-redundant genes, and KEGG analysis identified 8 significantly enriched cis-associated pathways and 152 significantly enriched trans-associated pathways (adjusted P&#x2009;<&#x2009;0.05), predominantly enriched for immune and inflammation-related pathways. These findings prioritized a subset of mastitis-associated lncRNAs for subsequent methylation and interaction-network analyses. A subset of these lncRNAs further overlapped differentially methylated regions (DMRs), suggesting a potential association between lncRNA expression changes and DNA methylation alterations. Integration of lncRNA-miRNA and miRNA-mRNA interactions identified lncRNA-miRNA-mRNA interaction networks involving DMR-associated lncRNAs. Among the prioritized candidates, MSTRG.28878.1 showed overlap with a hypomethylated promoter-associated DMR, increased expression, and multiple connections within the predicted interaction network. Together, these findings identify candidate lncRNAs, methylation-associated loci, and predicted molecular interactions associated with bovine subclinical mastitis and provide a resource for future functional investigation of candidate non-coding RNA-associated mechanisms in disease.

Animals

Genomic Insights into Mammaliicoccus sciuri from Subclinical Bovine Mastitis to Unveil Key Resistance, Virulence, Biofilm and Adaptation Traits.

The Mammaliicoccus sciuri (M. sciuri), is recognized as a reservoir of antimicrobial resistance (AMR) genes, poses challenges in the Indian dairy sector where antibiotic use is poorly regulated. This study aimed to genomically characterize M. sciuri (formerly Staphylococcus sciuri) isolates recovered from subclinical mastitis (SCM) cattle milk. A total of 128 composite (quarter-wise pooled) milk samples were collected from 199 households (HH) across 16 epiunits /villages in four blocks of Chikkaballapur district, Karnataka, India. Of these, 36 milk samples (28.13%, 36/128; 95% CI: 21.06&#x2013;36.46%) were diagnosed with SCM using the California Mastitis Test (CMT) and bacteriological culture yielded 113 isolates (88.28%; 113/128; 95% CI: 81.56&#x2013;92.77%) were phenotypically identified as Staph spp. Through molecular technique PCR targeting the gap gene, two isolates (1.77%; 2/113; 95% CI: 0.49&#x2013;6.22%) from Hosuru and Gattamaranahalli epiunits were confirmed as M. sciuri and both isolates were mecA-positives indicating methicillin resistance. Whole genome sequencing (WGS) identified 36&#x2013;37 resistance genes (mecA and blaZ), conferring resistance to &#x3b2;-lactams, macrolides, fluoroquinolones and aminoglycosides. Horizontal gene transfer (HGT) was evidenced by diverse mobile genetic elements (MGEs) such as SCCmec variants, insertion sequences, transposons (IS3, IS6, IS256, and IS1182) and plasmids (Rep1, Rep13, RepUS5 and RepUS43). Virulence profiling uncovered biofilm-associated genes (ica, bap) and heavy metal resistance operons (ars, cop, znu) suggesting mechanisms for environmental persistence and co-selection of resistance traits. Phylogenetic analysis of 99 global isolates revealed host-and geography-specific clustering with Indian isolates occupying distinct evolutionary niches. These findings highlights its possible role as an AMR reservoir and also in bovine mastitis.

Animals

Genomic and phenotypic characterization of mcr-9 carrying Enterobacter oligotrophicus recovered from bovine mastitis.

Bovine mastitis (BM) is a leading cause of economic loss in the dairy industry, driven by decreased milk yields, involuntary culling, and substantial veterinary costs. A single Gram-negative isolate recovered from BM was characterized in this study. For this, antimicrobial susceptibility testing (AST) was performed using the Neg-Urine-Combo 98 panel. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARG), virulence factors (VF) genes, plasmid replicons and prophage sequences. Comparative genomic analysis was performed through phylogenetic analysis. The BM isolate was phenotypically identified as Citrobacter rodentium, however, WGS analysis reclassified the isolate as Enterobacter oligotrophicus. Phenotypic AST revealed a resistance profile of 12%, with the isolate exhibiting resistance to &#x3b2;-lactams antimicrobials, specifically amoxicillin-clavulanate, ampicillin and the cephalosporin-cefoxitin. Conversely, susceptibility was demonstrated for the remaining tested antimicrobials. Genomic profiling identified 31 ARG, 10 VF genes and 6 prophage sequences within the E. oligotrophicus genome. This study provides the first evidence of E. oligotrophicus as a causative agent of BM, expanding the known spectrum of pathogens affecting the dairy industry by delivering the second complete genome of E. oligotrophicus available globally. The identification of 31 ARG, 10 VF, and 6 prophage sequences underscore the potential pathogenic risk and environmental resilience of this isolate. These findings highlight the critical role of WGS-based surveillance in identifying non-conventional mastitis pathogens and underscore the need for targeted mitigation strategies in veterinary medicine.

Animals

Whole genome sequence data set of methicillin-resistant Staphylococcus aureus isolated from a milkman associated with cows with subclinical mastitis in Kiruhura district, Uganda.

The whole-genome sequence data set for methicillin-resistant Staphylococcus aureus, which was isolated from a milkman associated with cows with subclinical mastitis in the Kiruhura district of Uganda, is presented here. The assembled genome size was 2822,509 bp, with a 33% GC, 2 Contigs, a Contig N50 of 2818,424, and 1 Contig L50. You can access the genome sequence and related metadata at https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_056782255.1/. This dataset can be used again for resistance gene mapping, comparing genomic analysis, and comprehending genetic diversity among MRSA isolates from Ugandan milkmen.

Antimicrobial-resistant genes Staphylococcus aureu

Treatment of clinical mastitis: two intramammary formulations compared.

Two hundred cases of mild clinical mastitis were treated on two farms using two intramammary preparations in quick release bases. One preparation contained penicillin and streptomycin, the other contained lincomycin, neomycin and prednisolone. Fifty-eight per cent of cases were clinically and bacteriologically cured using the first preparation; and 61 per cent of cases, with a similar range of organisms, using the second. The main infections were Streptococcus uberis and coliform; all but three of the infections were sensitive in vitro to one or both of the antibiotics in the preparations. On the farm with 100 cows, 69 per cent of cases were cured, while only 52 per cent were cured on the farm with 300 cows. It is suggested that it is more difficult to detect and treat cases in the larger herd. A number of reservations about the interpretation of the results are discussed.

Animals

[The interrelationship between the inhibitory activity of milk with different types of beta-lactoglobulins and the resistance of cattle to mastitis].

In the course of the investigation of piebald (black-white) cattle it is found that 17,62% animals produce the AA type beta-lactoglobulin, 49,52%--the AB type and 32,86%--the BB type. The higher inhibitory activity of milk with the BB type beta-lactoglobulin was found which retained in dilution 1 : 32. The total flora of teat, cyst and parenchyma milk of animals with the BB type beta-lactoglobulin as well as Enterococcus bacteria were much lower than in milk of cows with the AA type homogenous form. The animals with the first type of milk protein had mastitis more rarely as compared to those with the second type. The animals with heterogenous form of beta-lactoglobulin had intermediate values in most of their characteristics.

Animals

Ultrasound-Guided Microwave Ablation Versus Open Surgery for Benign Breast Disease: Impact on Postoperative Lactation.

BACKGROUND: Benign breast disease (BBD) is common in adult women. While ultrasound-guided microwave ablation (MWA) is an established minimally invasive treatment, its comparative impact on postoperative lactation, particularly mastitis, remains understudied. This study aimed to evaluate mastitis outcomes following MWA versus open surgery for BBD. METHODS: In this retrospective study, 79 patients (105 nodules) treated between January 2018 and August 2023 were included 41 patients (62 nodules) underwent MWA and 38 patients (43 nodules) underwent open surgery. All patients lactated within 5 years post-procedure. The primary outcome was the incidence of lactational mastitis without systemic symptoms (MWoSS) and mastitis with systemic symptoms (MSS). Patient satisfaction was also assessed. RESULTS: The incidence of MWoSS was significantly lower in the MWA group (19.5%) than in the open surgery group (50.0%). Similarly, MSS occurred in 4.9% of the MWA group versus 34.2% of the open surgery group. Multivariate analysis identified open surgery as an independent risk factor, associated with a 4.13-fold increased risk of MWoSS (OR = 4.125, 95% CI: 1.517-11.218) and a 10.14-fold increased risk of MSS (OR = 10.140, 95% CI: 2.107-48.793). Patient satisfaction was significantly higher in the MWA group (9.29 &#xb1; 0.72 vs. 8.15 &#xb1; 1.13, p < 0.001). CONCLUSION: Compared to open surgery, ultrasound-guided MWA for BBD is associated with a significantly lower risk of postoperative mastitis and higher patient satisfaction, supporting its use for women of reproductive age, particularly when future breastfeeding is planned.

Humans

[Diagnostic significance of lactose level of milk. 2. Lactose level in first quarter milk samples in various udder disorders].

The behaviour of lactose levels in first quarter milk was studied in the context of a large dairy herd in the presence of various udder health defects. The results were related to other criteria generally used in mastitis diagnosis. The lactose level in first quarter milk was found to be a diagnostic criterion which was properly applicable to detecting and identifying clinical and subclinical mastitis. A limit value of 4.6 per cent has been commonly assumed in literature and proved to be a sound basis for udder health assessment, provided that the given phase of lactation is taken into due consideration (exclusion of cows in the first month post partum as well as of aged milking cows from the seventh month of lactation). Also reported in this paper are results regarding milk lactose and its correlations with other criteria for mastitis diagnosis. A close correlation was found to exist between lactose level and electric conductivity of first quarter milk.

Animals

Genomic and food-safety evaluation of Staphylococcus chromogenes in Chinese dairy milk.

Non-aureus staphylococci and mammaliicocci (NASM) cause mastitis and may contaminate milk and dairy products. Milk samples (n&#xa0;=&#xa0;1916) from cows with subclinical or clinical mastitis (SCM and CM, respectively) were collected from 28 large-scale (> 500 lactating cows) Chinese dairy farms. Overall, 999 NASM isolates representing 19 species were identified by MALDI-TOF MS and cpn60 sequencing, with Staphylococcuschromogenes, Mammaliicoccus sciuri and Staphylococcus haemolyticus being most prevalent. Antimicrobial resistance (AMR) was determined with disc diffusion; non-susceptible to penicillin was most common (SCM, 30% and CM, 29%) whereas cefoxitin non-susceptible NASM accounted for 8-10% of isolates; among these, 12.5% carried mecA but none carried mecC. Galleria mellonella was used to assess virulence of 78 strains of S. chromogenes, a dominant species; subsequently, 32 strains, representing higher- and lower-virulence in the Galleria model, were selected for whole-genome sequencing and comparative genomics. S. chromogenes isolates from CM had higher virulence (p&#xa0;<&#xa0;0.05) than those from SCM. The 32 genomes comprised 20 sequence types, indicating high genetic diversity. No robust genomic marker of Galleria virulence phenotype was identified in this selected WGS subset. Acquired resistance genes (n&#xa0;=&#xa0;5) were detected, including a first report of fusC in S. chromogenes; the fusC-positive isolate had an elevated fusidic acid MIC (8&#xa0;mg/L). Although S. chromogenes persisted in milk at 4&#xa0;&#xb0;C, pasteurization (64&#xa0;&#xb0;C for 30&#xa0;min) reduced viable counts to below detection. This study provided new insights into the prevalence, AMR, genomic diversity, and dairy-chain relevance of milk-derived NASM, particularly S. chromogenes. However, the genomic findings were based on an intentionally selected WGS subset and should be interpreted as hypothesis-generating rather than population-representative.

Animals

Genomic diversity and resistance determinants of staphylococci from cow and buffalo milk.

BACKGROUND: Staphylococci are important mastitis pathogens in dairy animals and serve as reservoirs of antimicrobial resistance genes (ARGs) having zoonotic potential. Genomic characterization of resistant isolates is essential to understand their diversity, resistance mechanisms, and One Health implications. METHODS AND RESULTS: A total of 363 cow and buffalo milk samples-including 108 from animals with mastitis-were screened, yielding 98 staphylococcal isolates, comprising 20 Staphylococcus aureus and 78 coagulase-negative staphylococci (CoNS). Antimicrobial susceptibility testing revealed resistance to cefoxitin (CoNS: 21.7%; S. aureus: 10%), tetracycline (CoNS: 19.2%; S. aureus: 10%), erythromycin (CoNS:16.7%; S. aureus: 10%), gentamicin (CoNS: 10.2%; S. aureus: 10%) and fluoroquinolone (CoNS: 10.2%), while the majority were sensitive to chloramphenicol, cotrimoxazole (~&#x2009;95%, each), linezolid (~&#x2009;97%), and vancomycin (100%). Nineteen isolates, including two S. aureus, were cefoxitin-resistant, and eight carried the mecA gene. Whole genome sequencing of these eight isolates revealed genome sizes ranging from 2.27 to 2.78&#xa0;MB, with the methicillin resistant S. aureus (MRSA, ERSST98) isolate possessing the largest genome and the highest rRNA copy number. Comparative genomic analysis revealed various SCCmec types along with an extensive array of resistance determinants, encompassing aminoglycosides, macrolides, tetracyclines, efflux systems, and heavy metals, underscoring the multifaceted resistance repertoire of these strains. Virulence profiling of ERSST98 demonstrated a broad arsenal of adhesins, toxins, and biofilm&#x2011;associated genes, highlighting its pathogenic capacity. Mobile genetic elements with diverse plasmid replicons and insertion sequence families further contributed to genomic plasticity. CONCLUSIONS: Collectively, this study underscores the genomic diversity of methicillin-resistant staphylococci from dairy animals with extensive resistance determinants and highlights their zoonotic relevance within One Health framework.

Animals

Circulating levels of prolactin in human breast cancer.

Serum prolactin concentrations were measured by radioimmunoassays in 98 patients with established carcinoma of breast, 12 patients with cystic mastitis and 10 patients with gynaecomastia and compared with that of age matched normal control women. The serum prolactin levels in the patients with breast cancer, gynaecomastia or cystic mastitis were observed to be similar to that in normal women. It was interesting to note that the levels of prolactin in the luteal phase of the cycle were higher than that in the early follicular phase in normal women.

Adult

Isolation, identification, and genomic characterization of Staphylococcus aureus phage vB_SauL_202595 and its bacteriostatic application in dairy products.

Staphylococcus aureus is an important pathogen associated with bovine mastitis and dairy product contamination, posing economic and public health risks through the food chain. In this study, a temperate phage, vB_SauL_202595, was isolated from a dairy farm environmental sample using S. aureus SHZ-0127 as the host, and its biological characteristics, genomic features, and antibacterial activity in dairy matrices were evaluated. vB_SauL_202595 lysed 18 of 66 tested S. aureus strains, with a lysis susceptibility rate of 27.3%, including 5 highly susceptible strains, indicating a relatively limited host range. The optimal multiplicity of infection was 0.01, the latent period was approximately 30 min, and the burst size was approximately 316 PFU/cell. The phage remained stable at 4&#xb0;C-37&#xb0;C and pH 6-10. Genome analysis showed that vB_SauL_202595 belongs to the class Caudoviricetes, has a genome of 44,503 bp with 33.59% GC content, and encodes 63 predicted proteins. No typical antibiotic resistance genes or major virulence factors were detected; however, integrase and repressor genes were identified, supporting its temperate nature. vB_SauL_202595 inhibited S. aureus SHZ-0127 growth, reduced mature biofilm biomass, and decreased viable bacterial counts in milk and yogurt, with reductions of 1.23 and 1.42 log10 CFU/mL under representative conditions, respectively. From a One Health perspective, these findings provide foundational evidence for reducing S. aureus contamination and related antimicrobial resistance risks along the dairy chain. Overall, vB_SauL_202595 represents a candidate phage resource for dairy-associated S. aureus biocontrol research, but its limited host range and lysogeny-related genes require further safety assessment before food-related applications.IMPORTANCEStaphylococcus aureus is a major pathogen associated with bovine mastitis and a common contaminant in dairy products, causing economic losses and public health risks through the food chain. Although phage-based biocontrol has emerged as a promising strategy for controlling S. aureus contamination in dairy products, systematic evidence regarding phage activity in actual dairy matrices remains limited. In this study, we isolated and characterized a dairy farm environment-derived temperate phage, vB_SauL_202595, and evaluated its biological characteristics, genomic features, host range, stability, biofilm removal ability, and antibacterial performance in milk and yogurt. These findings provide foundational experimental evidence for phage-based dairy biocontrol against S. aureus. However, due to its limited host range and lysogeny-related genomic features, vB_SauL_202595 should be considered a candidate phage resource for further study. Broader validation, including phage-cocktail testing, long-term storage assays, product quality assessment, and regulatory safety evaluation, is needed before practical application.

Staphylococcus aureus

Genome-wide analysis of fitness determinants of Staphylococcus aureus during growth in milk.

Staphylococcus aureus is a major concern in the dairy industry due to its significance as a pathogen causing bovine mastitis as well as a source of food poisoning. The nutrient-rich milk environment supports bacterial growth, but the specific genetic determinants that facilitate S. aureus proliferation and persistence in milk are poorly understood. In this study, we conducted a genome-wide CRISPR interference sequencing (CRISPRi-seq) screen with the laboratory strain S. aureus NCTC8325-4, to identify fitness determinants essential for S. aureus growth and survival in milk. We identified 282 milk-essential genes, including those with key roles in DNA replication, protein synthesis, and metabolism. Comparative analysis with brain heart infusion (BHI) as growth medium, revealed 79 genes with differential fitness, highlighting specific adaptations required for growth in milk. Notably, we found that purine biosynthesis, folate cycle pathways, and metal acquisition were particularly important in this environment. Based on this, we further demonstrate that S. aureus is more sensitive to the folate inhibitors trimethoprim-sulfamethoxazole (TMP-SMX) in milk and identify several genes whose knockdown results in hypersensitivity to TMP-SMX in milk. Additionally, our analysis showed a relatively reduced importance of cell wall components, such as teichoic acids, for S. aureus fitness in milk, which is also reflected in reduced efficiency of antimicrobials targeting teichoic acids. Together, these findings provide new insights into the genetic basis of S. aureus fitness and antibiotic susceptibility in milk, offering directions for novel treatment strategies against bovine mastitis.

Animals

gamma-Glutamyltransferase in human and mouse breast tumours.

A series of experimental mouse tumours were assayed for their gamma-glutamyltransferase activities. Significantly raised activities were found in a transplantable spontaneous mammary carcinoma compared to normal or lactating mouse breast. A study was therefore undertaken of human breast tumours. Carcinomas showed significantly elevated enzyme levels when compared with normal tissue or histologically uninvolved tissue from a breast containing a carcinoma. Fibroadenoma and chronic mastitis also showed significantly elevated levels when compared with normal tissue and did not differ significantly from carcinoma tissue. Benign breast cyst fluid showed very high levels of enzyme activity. Binding properties of the enzyme to Con A-Sepharose suggested that while normal tissue and fibroadenomas contained only asialated enzyme, carcinomas, chronic mastitis and cyst fluid contained a substantial proportion of sialated enzyme.

Animals

Significance of fecal coliform-positive Klebsiella.

A total of 191 Klebsiella pneumoniae isolates of human clinical, bovine mastitis, and a wide variety of environmental sources were tested for fecal coliform (FC) response with the membrane filtration and most probable number techniques. Twenty-seven Escherichia coli cultures of human clinical and environmental origins were also tested. Eighty-five percent (49/58) of known pathogenic K. pneumoniae were FC positive, compared with 16% (19/120) of the environmental strains. E. coli results indicated 93% (13/14) of the clinical and 85% (11/13) of the environmental strains as FC positive. There was no significant difference in the incidence of FC-positive cultures between pathogenic Klebsiella and E. coli. pH measurements of K. pneumoniae and E. coli cultures growing in m-FC broth at 44.5 degrees C revealed three distinct pH ranges correlating with colony morphology. beta-Galactosidase assays of Klebsiella and E. coli cultures at 44.5 degrees C indicated all were able to hydrolyze lactose, even if they were FC negative by the membrane filtration or most probable number techniques. The FC response pattern appears stable in K. pneumoniae. Three pathogenic cultures showed no change in FC responses after 270 generations of growth in sterile pulp mill effluent. Since K. pneumoniae is carried in the gastrointestinal tract of humans and animals and 85% of the tested pathogenic strains were FC positive, the isolation of FC-positive Klebsiella organisms from the environment would indicate their fecal or clinical origin or both. The added fact that K. pneumoniae is an opportunistic pathogen of increasing importance makes the occurrence of FC-positive environmental Klebsiella, particularly in large numbers, a potential human and animal health hazard.

Animals