Inappropriate use of antibiotics exacerbates inflammation through OMV-induced pyroptosis in MDR Klebsiella pneumoniae infection.

The inappropriate use of antibiotics is a severe public health problem worldwide, contributing to the emergence of multidrug-resistant (MDR) bacteria. To explore the possible impacts of the inappropriate use of antibiotics on the immune system, we use Klebsiella pneumoniae (K. pneumoniae) infection as an example and show that imipenem increases the mortality of mice infected by MDR K. pneumoniae. Further studies demonstrate that imipenem enhances the secretion of outer membrane vesicles (OMVs) with significantly elevated presentation of GroEL, which promotes the phagocytosis of OMVs by macrophages that depends on the interaction between GroEL and its receptor, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). OMVs cause the pyroptosis of macrophages and the release of proinflammatory cytokines, which contribute to exacerbated inflammatory responses. We propose that the inappropriate use of antibiotics in the cases of infection by MDR bacteria such as K. pneumoniae might cause damaging inflammatory responses, which underlines the pernicious effects of inappropriate use of antibiotics.

Klebsiella pneumoniae infection causes mitochondrial damage and dysfunction in bovine mammary epithelial cells.

Klebsiella pneumoniae, an important cause of bovine mastitis worldwide, is strongly pathogenic to bovine mammary epithelial cells (bMECs). Our objective was to determine the role of mitochondrial damage in the pathogenicity of K. pneumoniae on bMECs, by assessing several classical indicators of mitochondrial dysfunction, as well as differentially expressed genes (DEGs). Two K. pneumoniae strains (HLJ-D2 and HB-AF5), isolated from cows with clinical mastitis (CM), were used to infect bMECs (MAC-T line) cultured in vitro. In whole-transcriptome analysis of bMECs at 6 h post-infection (hpi), there were 3453 up-regulated and 3470 down-regulated genes for HLJ-D2, whereas for HB-AF5, there were 2891 up-regulated and 3278 down-regulated genes (P < 0.05). Based on GO term enrichment of differentially expressed genes (DEGs), relative to the controls, the primary categories altered in K. pneumoniae-infected bMECs included cellular macromolecule metabolism, metabolic process, binding, molecular function, etc. Infections increased (P < 0.05) malondialdehyde concentrations and formation of reactive oxygen species in bMECs. Additionally, both bacterial strains decreased (P < 0.05) total antioxidant capacity in bMECs at 6 and 12 hpi. Furthermore, infections decreased (P < 0.05) mitochondrial membrane potential and increased (P < 0.01) mitochondrial calcium concentrations. Finally, severe mitochondrial swelling and vacuolation, as well as mitochondrial rupture and cristae degeneration, were detected in infected bMECs. In conclusion, K. pneumoniae infections induced profound mitochondrial damage and dysfunction in bMECs; we inferred that this caused cellular damage and contributes to the pathogenesis of K. pneumoniae-induced CM in dairy cows.

Serum iron concentration in cattle with endotoxaemia.

The objective of this study was to examine whether serum iron (Fe) concentration is useful as a prognostic biomarker for cows with acute coliform mastitis (ACM). Our study was composed of determining the reproducibility of serum Fe concentration as a prognostic criterion in cows with ACM (Study 1) and clarifying the sequential changes in serum Fe concentration in cattle that received endotoxin (Study 2). Seventy-seven cows with (n = 47) or without (n = 30) ACM were enrolled in Study 1. The proposed diagnostic cut-off value of serum Fe concentration indicating a poor prognosis of ACM based on the analysis of the receiver operating characteristic curves was < 31.5 µg/dL. Ten young cattle aged 176.8 ± 23.7 days were enrolled in Study 2. Five young cattle received endotoxin (LPS group) and the remaining five received physiological saline (control group). Blood collections were carried out before endotoxin challenge (pre), and 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the challenge. As a result, a significant decrease in serum Fe concentration was not observed until 24 h after endotoxin challenge. Because in cows with clinical ACM it is difficult to know the time course after infection, the alteration in serum Fe concentrations alone may be an insufficient prognostic criterion.

Molecular characterization of newly identified Klebsiella PKBSG14 and analysis of its effect on immune response and cell cycle progression using common catfish (Channa punctatus) as a model.

Microbial studies on Catfish revealed that Klebsiella is the most common pathogen causing prevalence of ulcers, fin erosion, and other lesions. During this study, a new strain of bacteria was isolated from Channa punctatus, and molecular identification by 16srRNA revealed the strain was Klebsiella PKBSG14 (Accession no KJ162158). The strain was also PCR positive for two virulent gene wcaG (Accession no LN606595) and rmpA (Accession no LN606594) responsible for inflammatory reactions and induction of innate immune response in the host cell. To study innate immune response induced by pathogenic infection the phagocytic interactive process between the spleen macrophages and KlebsiellaPKBSG14 was investigated using optical microscopy. FACS of splenic macrophages revealed that the phagocytic interaction leads to the process of macrophage cell cycle progression. A detailed study on the macrophage DNA content by performing DNA fragmentation and comet allowed us to study simultaneously host cell division as a function of phagocytosis and the findings unveiled the fact that Phagocytosis of KlebsiellaPKBSG14 aided in macrophage cell cycle progression but was less likely to complete mitosis. Here we also report the cytotoxic effect linked to the infection with KlebsiellaPKBSG14 by performing Cell viability assay, intracellular production of ROS, and mitochondrial transmembrane potential where it manifested itself in impaired cellular function. So, in summary, we simultaneously discovered a new strain of bacteria ie. Klebsiella PKBSG14 as well as deliberately attempted to study the immunomodulatory effect of isolated new stain on Channa punctatus by performing host-pathogen phagocytic interactive experiments, the cell cycle state of the host cell and pathogen-mediated cytotoxicity along with genotoxicity, and our results evidence a new immunomodulatory effect of KlebsiellaPKBSG14 infection on fish splenic macrophages.

Characterization of Klebsiella pneumoniae associated with cattle infections in southwest China using multi-locus sequence typing (MLST), antibiotic resistance and virulence-associated gene profile analysis

Abstract Klebsiella pneumoniae is important human and animal pathogen that causes a wide spectrum of infections. In this study, isolates from cattle nasal swabs samples were identified by 16S rRNA, and to evaluate the antimicrobial susceptibility, virulence gene carrying levels, and multilocus sequence typing of K. pneumoniae isolates. 33 isolates of K. pneumoniae were isolated and identified in 213 nasal swabs samples, of which 12 were hypervirulent K. pneumoniae strains. Extended Spectrum Beta-Lactamases genes were found in 93.4% of the strains. Of which, TEM was the most prevalent (93.4%), followed by CTX-M and SHV were 57.6% and 39.4%, respectively. A main mutation pattern of quinoloneresistance-determining region, Thr83-Ieu and Asp87-Asn in gyrA and Ser87-Ile in parC, was detected in 33 K. pneumoniae isolates. All the isolates harbored at least two virulence factor genes, with ureA (97.0%) and wabG (91.0%) exhibiting high carriage rates in 33 K. pneumoniae isolates. MLST revealed 7 sequence types, of which 3 STs (2541, 2581 and 2844) were newly assigned. Using eBURST, ST2844 and ST2541 were assigned to new clonal complex 2844. Our study provides evidence and biological characteristics of K. pneumoniae isolates from cattle upper respiratory tract in Southwest China.

Global Proteomics Revealed Klebsiella pneumoniae Induced Autophagy and Oxidative Stress in Caenorhabditis elegans by Inhibiting PI3K/AKT/mTOR Pathway during Infection.

The enterobacterium, Klebsiella pneumoniae invades the intestinal epithelium of humans by interfering with multiple host cell response. To uncover a system-level overview of host response during infection, we analyzed the global dynamics of protein profiling in Caenorhabditis elegans using quantitative proteomics approach. Comparison of protein samples of nematodes exposed to K. pneumoniae for 12, 24, and 36 h by 2DE revealed several changes in host proteome. A total of 266 host-encoded proteins were identified by 2DE MALDI-MS/MS and LC-MS/MS and the interacting partners of the identified proteins were predicted by STRING 10.0 analysis. In order to understand the interacting partners of regulatory proteins with similar or close pI ranges, a liquid IEF was performed and the isolated fractions containing proteins were identified by LC-MS/MS. Functional bioinformatics analysis on identified proteins deciphered that they were mostly related to the metabolism, dauer formation, apoptosis, endocytosis, signal transduction, translation, developmental, and reproduction process. Gene enrichment analysis suggested that the metabolic process as the most overrepresented pathway regulated against K. pneumoniae infection. The dauer-like formation in infected C. elegans along with intestinal atrophy and ROS during the physiological analysis indicated that the regulation of metabolic pathway is probably through the involvement of mTOR. Immunoblot analysis supported the above notion that the K. pneumoniae infection induced protein mis-folding in host by involving PI3Kinase/AKT-1/mTOR mediated pathway. Furthermore, the susceptibility of pdi-2, akt-1, and mTOR C. elegans mutants confirmed the role and involvement of PI3K/AKT/mTOR pathway in mediating protein mis-folding which appear to be translating the vulnerability of host defense toward K. pneumoniae infection.

First case report of non-human primates (Alouatta clamitans) with the hypervirulent Klebsiella pneumoniae serotype K1 strain ST 23: A possible emerging wildlife pathogen.

BACKGROUND: Hypervirulent strain of Klebsiella pneumoniae genotype K1 isolates have recently emerged, causing severe pyogenic liver abscess complicated by devastating metastatic infections in humans. METHODS: We describe a short outbreak of the non-human primate (NHP) research center, associated with a hypervirulent K. pneumoniae. The genetic similarity of the strains was evaluated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques, and virulence encoding genes were detected by polymerase chain reaction (PCR). RESULTS: The isolates were phenotypically like strains causing community-acquired invasive liver abscess syndrome in humans. All strains exhibited identical PFGE patterns and were found to belong to ST23 and presented a hypermucovisity phenotype and possessed magA and rmpA gene. CONCLUSION: This is the first case report of NHPs caused by K. pneumoniae displaying a hypermucoviscosity phenotype and belonging to capsular serotypes K1 and ST23.

Septicemia and Infection due to ESBL-producing K. pneumoniae Following Feline Renal Allograft Transplantation.

A 12 yr old castrated male domestic longhair underwent renal transplantation for treatment of chronic interstitial nephritis. Full-thickness intestinal biopsies obtained prior to transplantation revealed mild enteritis. Twelve months following transplantation, the patient underwent surgery for resection of a mesenteric mass causing septic peritonitis. The mesenteric mass was resected and an intestinal resection and anastomosis was performed. Extended-spectrum-ß-lactamase producing Klebsiella pneumoniae was cultured from the resected tissue and urinary tract. Bacterial rods were noted to be circulating in the bloodstream, causing septicemia. Despite aggressive treatment of the septic peritonitis and septicemia using surgical debridement, drain placement, aggressive antibiotic therapy with IV meropenem, and vasopressor support, the patient succumbed to persistent hypotension and suffered cardiopulmonary arrest. Extended-spectrum-ß-lactamase-producing bacteria are of growing concern in human and veterinary medicine, maintaining susceptibility often only to carbapenem and aminoglycoside antibiotics. Resistance to even those antibiotics is emerging. Veterinary patients with a history of antibiotic therapy, central venous or urinary catheterization, immunosuppression, enteric surgery, and an extended stay in the intensive care unit may be predisposed.

Seasonal shifts in bacteria associated with Jersey cows on a small dairy farm and the potential for bedding choice and low levels of iodine use to inhibit mastitic pathogens.

Milk products from small dairies are increasingly in demand, as access to pasture provides benefits to the cow, consumer, and environment. The productivity and profitability of small dairy farms particularly rely on the prevention of infectious diseases. Cattle on seasonal grazing dairies live primarily outdoors until inclement weather warrants relocation indoors. While shifts in the amounts of bacteria associated with livestock may be expected from this transition, potentially increasing risks for infectious diseases, changes in bacteria levels on cows relocated to indoor facilities have not been well-studied. In addition, the optimal use of bedding materials and iodine are critical in bovine infectious diseases prevention. However, the antibacterial potential of bedding material with high polyphenol content or low concentrations of iodine, are poorly understood. Cow teats were swabbed and total bacteria and coliform counts, as well as extracellular enzyme activities (EEA) were utilized to assess shifts in bacterial levels on cows at pasture and then housed indoors. To test the antibacterial efficacy of bedding materials, as well as low concentrations of povidone-iodine, growth curves with laboratory strains of Staphylococcus aureus and Klebsiella pneumoniae, as well as S. aureus isolated from a dairy farm, were performed with three concentrations of red cedar shavings or iodine. Post hoc multiple comparisons indicated that total bacteria, coliform, and ß-galactosidase activities were significantly greater among cows housed indoors compared to bacterial samples from cows at pasture. Laboratory strains of S. aureus, but not K. pneumoniae, were significantly inhibited by moderate and high treatments of red cedar shavings, while S. aureus isolated from a dairy were inhibited by the high treatment only. All low iodine concentrations significantly inhibited each bacterial strain investigated. These results may help optimize strategies for the prevention of infectious diseases of bovine udders critical to the productivity and profitability of small dairies.

Ischaemic distal limb necrosis and Klebsiella pneumoniae infection associated with arterial catheterisation in a cat.

This case report describes dorsal pedal arterial thrombosis and infection with Klebsiella pneumoniae subsequent to arterial catheter placement in a cat. The complication led to avascular necrosis of the metatarsal and pedal soft tissue. The catheter was placed for blood pressure monitoring during surgery for correction of a peritoneopericardial diaphragmatic hernia. The exact mechanism of thrombosis was unclear. Amputation of the limb was required and the histopathological findings are presented. This is the first report of such a complication.

Changes in acute-phase proteins and cytokines in serum and milk whey from dairy cows with naturally occurring peracute mastitis caused by Klebsiella pneumoniae and the relationship to clinical outcome.

This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α(1)-acid glycoprotein and IL-1ß in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 µg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.

Double ureters with ureteral ectopia in a domestic shorthair cat.

A 5-month-old domestic shorthair cat was presented for evaluation of urinary incontinence since birth. Excretory urography revealed dilated double ectopic ureters draining a hydronephrotic right kidney. Urine culture yielded a pure culture of Klebsiella pneumoniae. The cat was treated initially with bactericidal antibiotics, followed by right-sided nephroureterectomy. The surgery and antibiotic therapy led to complete resolution of urinary incontinence and urinary tract infection. Ureteral duplication is an unusual congenital anomaly that has not been previously reported in the cat.

Multidrug-resistant bacterial ingluvitis associated with squamous cell carcinoma in a budgerigar (Melopsittacus undulatus).

A 2-year-old budgerigar (Melopsittacus undulatus) was presented to the Massey University Veterinary Teaching Hospital for chronic regurgitation. Multiple drug-resistant Klebsiella oxytoca was cultured from the crop. Necropsy revealed a mass in the crop that was histologically diagnosed as squamous cell carcinoma (SCC). To the authors' knowledge, this is the first report of SCC in a budgerigar associated with a multidrug-resistant bacterial ingluvitis.

Characterization of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae.

Gram-negative bacteria are responsible for almost one-half of the clinical cases of mastitis that occur annually. Of those gram-negative bacteria that induce mastitis, Klebsiella pneumoniae remains one of the most prevalent. Detection of infectious pathogens and the induction of a proinflammatory response are critical components of host innate immunity. The objective of the current study was to characterize several elements of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae. The inflammatory cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), 2 proteins that contribute to host recognition of gram-negative bacteria, were studied. The contralateral quarters of 7 late-lactating Holstein cows were challenged with either saline or K. pneumoniae, and milk and blood samples were collected. Initial increases in the chemoattractants C5a and IL-8, as well as TNF-alpha, were evident in infected quarters within 16 h of challenge and were temporally coincident with increases in milk somatic cells. Augmented levels of TNF-alpha and IL-8 were observed in infected quarters until >48 h postchallenge, respectively. Elevated levels of IL-12, IFN-gamma, and the antiinflammatory cytokine, IL-10, which were first detected between 12 and 20 h postinfection, persisted in infected quarters throughout the study (>96 h). Initial increases in milk LBP and sCD14 were detected 16 and 20 h, respectively, after challenge. Together, these data demonstrate that intramammary infection with K. pneumoniae elicits a host response characterized by the induction of proinflammatory cytokines and elevation of accessory molecules involved in LPS recognition.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities in the sera and milk of cows with naturally occurring coliform mastitis.

The presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities was determined in milk and serum of cows with naturally occurring coliform mastitis (CFM). TNF-alpha was detected in the sera from 26 of 32 cows with CFM. TNF-alpha levels were higher in the sera than in the milk. IL-6 was high in the sera of surviving CFM animals, but was low in animals that died and in healthy controls. Furthermore, the mean level of IL-6 was 20-fold higher in the milk than in the sera of mastitic cows. The level of IL-6 in the serum was correlated to that in the milk in individual animals. The presence of IL-6 and TNF-alpha in the sera appears to relate to severe clinical condition of CFM, in the milk whereas they may play a role in generating inflammation of the mammary gland.

Intracapsular prostatic omentalization: a new technique for management of prostatic abscesses in dogs.

Twenty dogs with abscessation of the prostate gland were treated by celiotomy, drainage, and digital exploration of the gland through bilateral capsulectomy wounds. Omentum was introduced through the capsulectomy wounds and packed into the abscess cavities around the prostatic urethra. Most dogs were discharged from the hospital within 48 hours of the surgery. Long-term resolution of the problem (no recurrence within 12 months) was achieved in 19 dogs. One dog had recurrent abscessation that was managed by the placement of dependent Penrose drains. Temporary tube cystostomy was performed postoperatively in one dog for the management of transient dysuria. One dog with septic peritonitis resulting from preoperative abscess rupture was managed by concurrent open peritoneal drainage. Intracapsular prostatic omentalization was a simple and effective means of managing prostatic abscessation with minimal requirement for postoperative hospitalization of the patient. The frequency of serious complications, including postoperative urinary incontinence, was low.

Resolution of a left ureteral stone using electrohydraulic lithotripsy in a thoroughbred colt.

A 3-year-old Thoroughbred colt was presented for evaluation of azotemia and anorexia. Physical examination revealed a ureterolith in the left ureter, approximately 10 cm from the bladder, which was thought to obstruct urine flow by approximately 90% when viewed cystoscopically. Ultrasonographic examination of both kidneys revealed indistinct corticomedullary junctions, and the right kidney was more hyperechoic. A percutaneous biopsy of the right kidney revealed chronic interstitial nephritis with marked interstitial medullary fibrosis. Medical therapy consisting of IV fluids, sodium chloride PO, and ammonium chloride PO was initiated. Ureteroscopic electrohydraulic lithotripsy via a perineal urethrostomy was used to successfully remove the stone. Klebsiella oxytoca, which responded to oral enrofloxacin therapy, was cultured from the urine after surgery. Azotemia resolved and the horse resumed training.

Septic cholangiohepatitis and cholangiocarcinoma in a horse.

Septic cholangiohepatitis was diagnosed in an 11-year-old Warmblood gelding with a history of intermittent colic and fever. Klebsiella pneumoniae, susceptible to gentamicin, was cultured from the biopsy specimen. However, treatment with gentamicin was unsuccessful, and histologic examination and bacteriologic culture of a biopsy specimen obtained 3 weeks later revealed progression of the hepatic inflammation and yielded growth of gentamicin-resistant K pneumoniae. At this time, several discrete hyperechoic structures, suggestive of biliary calculi, were seen ultrasonographically. A change in antibiotic treatment was associated with gradual resolution of clinical signs. Five months after initial examination, the horse had a sudden onset of severe right forelimb lameness. The horse responded to treatment with antibiotics and phenylbutazone, but lameness and fever that was unresponsive to treatment recurred 7 months later, and the horse was euthanatized. Necropsy revealed nodules throughout the liver and a mass associated with the right metacarpophalangeal joint. Histologic and immunohistochemical examination revealed carcinomatous infiltration of the liver and metacarpophalangeal joint. The tumor was probably of biliary origin. Carcinoma should be considered in cases of septic cholangiohepatitis unresponsive to antibiotic treatment.