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ABSTRACT Bacterial isolates from the human urinary microbiome have been extensively studied for their antibiotic resistance; however, little work has been done on those isolates that are difficult to grow in vitro. This study was designed to qualify a serum-based medium, New York City Broth III (NYCIII), and a broth microdilution method to determine the antibiotic susceptibility of previously underreported or undescribed microbes that have a difficult time growing in standard Mueller-Hinton broth. Here, we demonstrate that NYCIII microbroth dilution can be an effective method for the determination of antibiotic susceptibility of species found in the human urinary microbiome. We show that this method serves well to characterize fastidious and anaerobic urinary microbes that have no Clinical and Laboratory Standards Institute (CLSI) guidelines, including several in the families Aerococcaceae, Lactobacillaceae, or Actinomycetaceae. Previous studies using expanded quantitative urine culture reveal that urine samples from clinical patients are commonly polymicrobial in composition. Thus, we test whether NYCIII can serve as a viable harmonized medium, capable of supporting antibiotic susceptibility testing in a range of fastidious, non-fastidious, and anaerobic urinary microbes. We propose this methodology to be standardized comparable to CLSI standards to allow for resistance testing in uncharacterized urinary bacteria.IMPORTANCEAntibiotic susceptibilities of fastidious and anaerobic bacteria of the human urinary microbiome are largely underreported due to difficulty in growing them in the lab environment. The current standard medium, Muller-Hinton broth, has difficulty supporting the growth of many of these species, leaving microbiologists without a standardized method. To address this need, this study offers a methodology to survey susceptibilities in a high-throughput manner of these understudied microbes with a proposed harmonized medium, NYCIII, which is capable of supporting the growth of both fastidious and non-fastidious urinary microbes. Broader standardization of this method can allow for the development of antibiotic-resistant breakpoints of the many uncharacterized urinary microbes.

The advent of sensitive enhanced culture (metaculturomic) and culture-independent DNA-based (metagenomic) methods has revealed a rich collection of microbial species that inhabit the human urinary tract. Known as the urinary microbiome, this community of microbes consists of hundreds of distinct species that range across the entire phylogenetic spectrum. This new knowledge clashes with standard clinical microbiology laboratory methods, established more than 60 years ago, that focus attention on a relatively small subset of universally acknowledged uropathogens. Increasing reports support the hypothesis that this focus is too narrow. Single uropathogen reports are common in women with recurrent urinary tract infection (UTI), although wider disruption of their urinary microbiome is likely. Typical “UTI” symptoms occur in patients with “no growth” reported from standard culture and sometimes antibiotics improve these symptoms. Metaculturomic and metagenomic methods have repeatedly detected fastidious, slow growing, and/or anaerobic microbes that are not detected by the standard test in urine samples of patients with lower urinary tract symptoms. Many of these microbes are also detected in serious non-urinary tract infections, providing evidence that they can be opportunistic pathogens. In this review, we present a set of poorly understood, emerging, and suspected uropathogens. The goal is to stimulate research into the biology of these microbes with a focus on their life as commensals and their transition into pathogens

The discovery of the urinary microbiome (urobiome) has created opportunities for urinary health researchers who study a wide variety of human health conditions. This manuscript describes an analysis of catheterized urine samples obtained from 1,004 urobiome study participants with the goal of identifying the most abundant and/or prevalent (common) taxa in five clinically relevant cohorts: unaffected adult women (n=346, 34.6%), urgency urinary incontinence (UUI) (n=255, 25.5%), stress urinary incontinence (SUI) (n=50, 5.0%), urinary tract infection (UTI) (n=304, 30.4%), and interstitial cystitis/painful bladder syndrome (IC/PBS) (n=49, 4.9%). Urine was collected via transurethral catheter and assessed for microbes with the Expanded Quantitative Urine Culture (EQUC) technique. For this combined analytic cohort, the mean age was 59 ± 16; most were Caucasian (n=704, 70.2%), Black (n=137, 13.7%), or Hispanic (n=130, 13.0%), and the mean BMI was 30.4 ± 7.7. Whereas many control or IC/PBS cohort members were EQUC-negative (42.4% and 39.8%, respectively), members of the other 3 cohorts were extremely likely to have detectable microbes. The detected urobiomes of the controls and IC/PBS did not differ by alpha diversity or genus level composition and differed by only a few species. The other 3 cohorts differed significantly from the controls. As expected, Escherichia was both prevalent and highly abundant in the UTI cohort, but other taxa also were prevalent at more moderate abundances, including members of the genera Lactobacillus, Streptococcus, Staphylococcus, Corynebacterium, Actinomyces, and Aerococcus. Members of these genera were also prevalent and highly abundant in members of the UUI cohort, especially Streptococcus anginosus. Intriguingly, these taxa were also detected in controls but at vastly lower levels of both prevalence and abundance, suggesting the possibility that UUI-associated symptoms could be the result of an overabundance of typical urobiome constituents. Finally, prevalence and abundance of microbes in the SUI cohort were intermediate to those of the UUI and control cohorts.These observations can inform the next decade of urobiome research, with the goal of clarifying the mechanisms of urobiome community composition and function. There is tremendous potential to improve diagnosis, evaluation and treatment for individuals affected with a wide variety of urinary tract disorders.

An N-balance experiment was conducted to test the hypothesis that d-Methionine (d-Met) has the same bioavailability and efficacy as l-Methionine (l-Met) when fed to weanling pigs. A Met-deficient basal diet containing 0.24% standardized ileal digestible (SID) Met was formulated. Six additional diets were formulated by adding 0.036%, 0.072%, or 0.108% d-Met or l-Met to the basal diet, and these diets, therefore, contained 77%, 87%, or 97% of the requirement for SID Met. Fifty-six barrows (10.53 ± 1.17 kg) were housed in metabolism crates and allotted to the seven diets with eight replicate pigs per diet. Feces and urine were collected quantitatively with 7-d adaptation and 5-d collection periods. Blood and tissue samples from pigs fed the basal diet and pigs fed diets containing 0.108% supplemental Met were collected on the last day. Results indicated that N retention (%) linearly increased (P < 0.01) as supplemental d-Met or l-Met increased in diets. Based on N retention (%) as a response, the linear slope-ratio regression estimated the bioavailability of d-Met relative to l-Met to be 101% (95% confidence interval: 57%-146%). The villus height and crypt depth in the jejunum were not affected by the Met level or Met source. Total antioxidant capacity or thiobarbituric acid reactive substance concentrations in plasma or tissue samples from pigs fed the control diet or diets containing 0.108% supplemental d-Met or l-Met were not different. Abundance of mRNA for some AA transporters analyzed in intestinal mucosa of pigs also did not differ. Therefore, it is concluded that d-Met and l-Met are equally bioavailable for weanling pigs.

The objective of this experiment was to investigate the effects of dietary crude protein (CP) content and crystalline amino acids (CAA) supplementation patterns on the histomorphology, immune response, and microbial metabolites in weaned pigs under clean (CSC) or unclean sanitary conditions (USC). One hundred forty-four piglets (6.35 ± 0.63 kg BW) were assigned to 1 of 3 diets: a high CP (HCP; 21%) and two low CP (LCP; 18%) diets supplemented with 10 crystalline indispensable amino acids (IAA) to meet all IAA requirements or only 6 IAA (Lys, Met, Thr, Trp, Val, and Ile) to meet IAA requirements except for Leu, His, and Phe. Each treatment had 8 replicates per sanitary condition with 3 pigs per pen. The CSC room was cleaned and washed weekly, whereas the USC room remained unwashed throughout the experiment and had sow manure spread in the pens. Data were analyzed using the MIXED procedure of SAS with orthogonal contrasts. Pigs fed the LCP diets had longer (P < 0.05) jejunal villi and higher villus height to crypt depth ratios than those fed the HCP diet. No significant difference is found in histomorphology between pigs fed LCP diets with different CAA supplementation patterns. Pigs raised under USC had greater (P < 0.05) plasma interleukin (IL)-10 and IL-6 concentrations and reduced (P < 0.05) plasma tumor necrosis factor-alpha concentration. Also, the LCP diets resulted in a greater (P < 0.05) plasma IL-10 concentration. Interactions (P < 0.05) between sanitation and dietary CP content were observed for goblet cell density and acetate and total volatile fatty acids contents in the jejunum. In conclusion, CAA supplementation patterns did not affect gut morphology in weaned pigs. Feeding LCP diets to weaned pigs improved gut morphology and microbial metabolites in jejunum under USC and ameliorated systemic inflammation induced by USC. [ABSTRACT FROM AUTHOR]

An experiment was conducted to evaluate the effect of feeding low crude protein (CP) diets and supplying 110% of the first 5 limiting AA on performance and diarrhea incidence in weaned pigs. One of 3 treatments were randomly assigned to 81 piglets (BW: 8.2 ± 1.2 kg; 9 pens/treatment; 3 pigs/pen) during 2 phases (3 wk each): a high CP (HP; 21 and 18%), a low CP (LP; 18 and 15%), or a low CP at 110% Lys, Met, Trp, Thr, Val requirements (LPAA; 18 and 15%) diet for phase 1 and 2, respectively. Pigs had ad libitum access to feed and water. Data were analyzed using PROC MIXED of SAS. During phase 1, pigs fed HP had the greatest ADG (P < 0.05), but ADFI and G:F were not different among treatments. In phase 2, no effect of treatments was observed on growth performance. Overall, pigs fed LPAA had the lowest (P < 0.05) ADG and G:F, and feeding LP decreased (P < 0.05) G:F. The ADFI was not affected by the treatments. Only during wk 3, pigs fed LPAA increased (P < 0.05) and LP tended to increase (P < 0.10) frequency of diarrhea compared to HP. On d 21, no treatment effect was observed for ileal villi height, but LPAA reduced (P < 0.05) and LP tended to reduce (P < 0.10) crypt depth compared to HP. Diet analysis revealed that LP was deficient in Ile and non-essential AA (NEAA), and LPAA contained inadequate levels of Ile, Leu, His and NEAA, which explains why reduced pig performance was achieved with these 2 diets. In conclusion, low CP diets, even with additional supplementation of the first 5 limiting AA, if deficient in the next limiting AA, results in negative effects on performance and diarrhea incidence. [ABSTRACT FROM AUTHOR]