In vitro synergistic activity of NCL195 in combination with colistin against Gram-negative bacterial pathogens.

In this study, the potential of using the novel antibiotic NCL195 combined with subinhibitory concentrations of colistin against infections caused by Gram-negative bacteria (GNB) was investigated. We showed synergistic activity of the combination NCL195 + colistin against clinical multidrug-resistant GNB pathogens with minimum inhibitory concentrations (MICs) for NCL195 ranging from 0.5-4 µg/mL for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, whereas NCL195 alone had no activity. Transmission electron microscopy of the membrane morphology of E. coli and P. aeruginosa after single colistin or combination drug treatment showed marked ultrastructural changes most frequently in the cell envelope. Exposure to NCL195 alone did not show any change compared with untreated control cells, whereas treatment with the NCL195 + colistin combination caused more damage than colistin alone. Direct evidence for this interaction was demonstrated by fluorescence-based membrane potential measurements. We conclude that the synergistic antimicrobial activity of the combination NCL195 + colistin against GNB pathogens warrants further exploration for specific treatment of acute GNB infections.

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations.

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P < 0.0001), and many provirus-positive southern animals failed to express any detectable KoRV RNA. Across both populations there was a positive association between proviral load and neoplasia (P = 0.009). Potential reasons for the differences in the nature of KoRV infection between the two populations are discussed.

Antimicrobial activity of thyme oil, oregano oil, thymol and carvacrol against sensitive and resistant microbial isolates from dogs with otitis externa.

BACKGROUND: Multidrug-resistant pathogens present a major global challenge in antimicrobial therapy and frequently complicate otitis externa in dogs. HYPOTHESIS/OBJECTIVES: In vitro efficacy of oregano oil, thyme oil and their main phenolic constituents against bacterial and fungal isolates associated with canine otitis externa were investigated. It was hypothesized that the main phenolic components would have greater antimicrobial activity compared to the relative essential oil. METHODS AND MATERIALS: Antimicrobial susceptibility testing was performed using broth microdilution with spot-plating technique to determine minimum inhibitory and bactericidal/fungicidal concentrations (MICs, MBCs and MFCs). A time-kill kinetics assay was performed to confirm the bactericidal and fungicidal activity of the oils and their phenolic constituents. One hundred bacterial and fungal isolates, including meticillin-susceptible Staphylococcus pseudintermedius (n = 10), meticillin-resistant S. pseudintermedius (n = 10), ß-haemolytic Streptococcus spp. (n = 20), Pseudomonas aeruginosa (n = 20; including 10 isolates resistant to one or two antimicrobials), Proteus mirabilis (n = 20) and Malassezia pachydermatis (n = 20) from dogs with otitis externa were used. RESULTS: Oregano oil, thyme oil, carvacrol and thymol exhibited antibacterial activity against all bacterial and fungal isolates tested. MIC90 values ranged from 0.015 to 0.03% (146-292 µg/mL) for the Gram-positive bacteria and P. mirabilis. For P. aeruginosa and M. pachydermatis, MIC90 values ranged from 0.09 to 0.25% (800-2,292 µg/mL). CONCLUSIONS AND CLINICAL SIGNIFICANCE: Oregano oil, thyme oil, carvacrol and thymol showed good in vitro bactericidal and fungicidal activity against 100 isolates from dogs with otitis externa, including some highly drug-resistant isolates. These essential oils and their main phenolic constituents have the potential to be further investigated in vivo for the treatment of canine otitis externa.

Biofilm production by pathogens associated with canine otitis externa, and the antibiofilm activity of ionophores and antimicrobial adjuvants.

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N-acetylcysteine (NAC), Tris-EDTA and disodium EDTA) were investigated spectrophotometrically (OD570nm ) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000-10,000 µg/ml and from 20,000-80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris-EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16-fold and eightfold higher than the MIC/MBC of NAC and Tris-EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris-EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm-associated OE in dogs.

Prevalence and clinical significance of koala retrovirus in two South Australian koala (Phascolarctos cinereus) populations.

PURPOSE: Koala retrovirus (KoRV-A) is 100  % prevalent in northern Australian (Queensland and New South Wales) koala populations, where KoRV-B has been associated with Chlamydia pecorum disease and the development of lymphosarcoma. In southern populations (Victoria and South Australia), KoRV-A is less prevalent and KoRV-B has not been detected in Victoria, while the current prevalence in South Australian populations is unknown but is thought to be low. This study aimed to determine (i) the prevalence of KoRV in the two largest South Australian koala populations [Kangaroo Island (KI) and Mount Lofty Ranges (MLR)], (ii) KoRV subtype and (iii) if an association between KoRV and C. pecorum exists. METHODOLOGY: Wild koalas were sampled in KI ( n =170) between 2014 and 2017 and in MLR ( n =75) in 2016. Clinical examinations were performed, with blood collected for KoRV detection and typing by PCR. RESULTS: KoRV prevalence was 42.4  % [72/170, 95 % confidence interval (CI): 34.9-49.8  %] in KI and 65.3  % (49/75, 95 % CI: 54.6-76.1  %) in MLR. Only KoRV-A, and not KoRV-B, was detected in both populations. In MLR, there was no statistical association between KoRV and C. pecorum infection (P =0.740), or KoRV and C. pecorum disease status ( P=0.274), although KoRV-infected koalas were more likely to present with overt C. pecorum disease than subclinical infection (odds ratio: 3.15, 95 % CI: 0.91-5.39). CONCLUSION: KoRV-A is a prevalent pathogen in wild South Australian koala populations. Future studies should continue to investigate KoRV and C. pecorum associations, as the relationship is likely to be complex and to differ between the northern and southern populations.

In vitro antimicrobial activity of seven adjuvants against common pathogens associated with canine otitis externa.

BACKGROUND: An antibiotic adjuvant is a chemical substance used to modify or augment the effectiveness of primary antimicrobial agents against drug-resistant micro-organisms. Its use provides an alternative approach to address the global issue of antimicrobial resistance and enhance antimicrobial stewardship. HYPOTHESIS/OBJECTIVES: To determine the antimicrobial activity of a panel of potential antimicrobial adjuvants against common pathogens associated with canine otitis externa (OE). ANIMALS/ISOLATES: A number of type strains and clinical isolates (n = 110) from canine OE were tested including Staphylococcus pseudintermedius, ß-haemolytic Streptococcus spp., Pseudomonas aeruginosa, Proteus mirabilis and Malassezia pachydermatis. METHODS AND MATERIALS: Antimicrobial activities of monolaurin, monocaprin, N-acetylcysteine (NAC), polymyxin B nonapeptide, Tris-EDTA, Tris-HCL and disodium EDTA were tested using microdilution methodology according to CLSI guidelines. RESULTS: N-acetylcysteine, Tris-EDTA and disodium EDTA had antimicrobial activity against both type strains and otic pathogens. The other adjuvants tested had limited to no efficacy. NAC had a minimal inhibitory concentration (MIC) range of 2,500-10,000 µg/mL for the various organisms. Pseudomonas aeruginosa isolates were eight times more susceptible to disodium EDTA in the presence of Tris-HCL in comparison to disodium EDTA alone. Malassezia pachydermatis isolates were most susceptible to Tris-EDTA (MIC90  = 190/60 µg/mL) and disodium EDTA (MIC90  = 120 µg/mL). CONCLUSIONS AND CLINICAL RELEVANCE: N-acetylcysteine, Tris-EDTA and disodium EDTA have intrinsic antimicrobial activity and represent promising adjuvants that could be used to enhance the efficacy of existing antibiotics against Gram-negative and multidrug-resistant bacterial infections. These agents could be combined with other antimicrobial agents in a multimodal approach for mixed ear infections in dogs.

Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle.

BACKGROUND: Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis. METHODS: Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis. RESULTS: Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5-8 µg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21-35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%). CONCLUSION: Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows.

Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli.

BACKGROUND: Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS: Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS: Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS: Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Occult bacterial lower urinary tract infections in cats-urinalysis and culture findings.

Bacterial urinary tract infections (UTIs) can be detected in feline urine submitted for urinalysis and culture as part of the diagnostic workup for a variety of conditions. Our aim was to investigate urinalysis and culture findings in urine specimens from cats with no history of lower urinary tract signs. Study inclusion criteria required cystocentesis specimens from cats with no history of lower urinary tract signs, inappropriate urination, or previous UTI (including pyelonephritis). Of 132 specimens, 38 were culture positive and 94 were culture negative. Culture positive urine specimens were more likely to come from older female cats (p=0.03, p<0.001, respectively) and they had higher pH (p=0.001), erythrocyte (p=0.013) and leukocyte counts (p=0.003) than culture negative urine specimens. Gram-negative infected specimens (n=15) had lower urine specific gravity and higher leukocyte counts than Gram-positive infected specimens (n=21; p=0.0012, p=0.005, respectively) and culture negative specimens (p=0.003, p<0.0001, respectively). Urine protein:creatinine ratio was higher in Gram-negative infected urine than in culture negative urine (p=0.013). Enterococcus faecalis was the most commonly isolated bacteria (19 of a total of 44 isolates; 43.2%) and E. coli phylogenetic group B2 was the most common Gram-negative isolate (14 of a total of 44 isolates; 31.8%). We conclude that feline bacterial urinary tract infections can occur in cats without lower urinary tract signs, particularly older females and that they are associated with high urine erythrocyte and leukocyte counts.

Trade-off between iron uptake and protection against oxidative stress: deletion of cueO promotes uropathogenic Escherichia coli virulence in a mouse model of urinary tract infection.

The periplasmic multicopper oxidase (CueO) is involved in copper homeostasis and protection against oxidative stress. Here, we show that the deletion of cueO in uropathogenic Escherichia coli increases its colonization of the urinary tract despite its increased sensitivity to hydrogen peroxide. The cueO deletion mutant accumulated iron with increased efficiency compared to its parent strain; this may account for its advantage in the iron-limited environment of the urinary tract.

Prevalence of bacterial species in cats with clinical signs of lower urinary tract disease: recognition of Staphylococcus felis as a possible feline urinary tract pathogen.

This study investigated the prevalence of bacterial pathogens of the urinary tract in Australian cats. Urine was collected by cystocentesis and subjected to urinalysis, bacterial culture and susceptibility testing. A total of 126 isolates were obtained from 107 culture-positive cats. Escherichia coli was most commonly isolated (37.3% of isolates) with the majority of isolates showing susceptibility to the 14 antimicrobials tested. Just over a quarter of isolates (27.0%) were Enterococcus faecalis, which showed resistance to cephalosporins and clindamycin. Staphylococcus felis, a previously unreported feline urinary tract pathogen which was susceptible to all antimicrobial agents tested, comprised 19.8% of the isolates. S. felis was significantly associated with urine that had a higher specific gravity (p=0.011) and pH (p=0.006) and was more likely to contain crystals (p=0.002) than urine from which other bacterial species were isolated. This is the first published study that associates the isolation of S. felis with clinical signs of lower urinary tract disease in cats.

Characterization of Treponema phagedenis-like spirochetes isolated from papillomatous digital dermatitis lesions in dairy cattle.

Four spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar to Treponema phagedenis and 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity among T. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.