Standard Aerobic Culture Research Articles

MSMEG_0918 is not Essential for the Growth of Mycobacteriumsmegmatis.

Copper homeostasis plays a crucial role in mycobacteria. In Mycobacterium tuberculosis , Rv0474 is a copper-responsive regulator with a copper-binding motif but its homolog in Mycobacterium smegmatis , MSMEG_0918, lacks the copper-binding motif. We generated MSMEG_0918 knockdown strains of M. smegmatis using CRISPRi. We confirmed the strains had varying levels of MSMEG_0918 expression using RT-PCR. We demonstrated that MSMEG_0918 under-expression did not alter the growth of M. smegmatis in standard aerobic culture as compared to the wild-type. Our knockdown strains (CHROME1 and CHROME2) could be further used towards understanding the role of MSMEG_0918 in M. smegmatis .

Association between quarter-level milk somatic cell count and intramammary bacterial infection in late-lactation Irish grazing dairy cows

The objectives of our study were to describe quarter-level prevalence of intrammamary infection (IMI), to evaluate the performance of commonly used somatic cell count (SCC) thresholds for the diagnosis of quarter-level IMI, and to determine those with maximized sensitivity (Se) and specificity (Sp) for identifying quarter-level IMI as defined by positive aerobic culture in late-lactation grazing dairy cows. In this observational study, quarter milk samples were collected from all cows in 21 commercial spring-calving, pasture-based Irish dairy herds. Total SCC determination and aerobic bacterial culture were performed in 8,177 quarter milk samples obtained between 238 and 268 d in milk from 465 primiparous and 1,609 multiparous cows. The Se and Sp of SCC thresholds used for diagnosis of IMI were evaluated against the gold standard aerobic culture separately for all, primiparous, and multiparous cows. The overall prevalence of bacteriologically infected quarters was 6.3%, and it was higher among primiparous (11.3%) than multiparous cows (5.5%). However, considering all samples, quarter-level SCC was higher for multiparous than for primiparous cows (195,250 ± 21,422 vs. 115,940 ± 26,260 cells/mL). Associated Se and Sp for the 200,000 cells/mL threshold were 59.2% and 88.0% for all, 52.7% and 95.4% for primiparous, and 62.9% and 85.9% for multiparous cows, respectively. Receiver operating characteristic curve analyses determined the thresholds that optimized the Se and Sp of a positive bacterial culture: 101,000 cells/mL for all cows [Se = 80.0%; Sp = 76.4%; area under the curve (AUC) = 0.84], 61,000 cells/mL for primiparous (Se = 87.1%; Sp = 84.0%; AUC = 0.90), and 101,000 cells/mL for multiparous (Se = 80.9%; Sp = 72.6%; AUC = 0.83). The results indicate that the 200,000 cells/mL threshold was inefficient in identifying late-lactation quarter-level IMI (low Se) in the studied herds where the main etiological agent was Staphylococcus aureus. Suggested quarter-level SCC thresholds have the potential of serving as a supporting tool for dry cow therapy decisions and warrant further study in late-lactation cows from spring-calving, pasture-based herds with S. aureus as the main pathogen causing IMI.

Septic Arthritis and Granulomatous Osteomyelitis in a Bearded Dragon (Pogona vitticeps) Caused by Mycobacterium sp.

A 3-yr-old female bearded dragon (Pogona vitticeps) was presented to the Exotic Pet and Wildlife Veterinary Teaching Hospital at the National Autonomous University of Mexico with a 3-day history of right hindlimb swelling. Radiographs revealed lysis of the right stifle and tarsal bone. Fine-needle aspiration from the stifle revealed large numbers of macrophages and giant cells, and Ziehl-Neelsen stain revealed intracytoplasmic acid-fast bacilli-like bacteria. Standard aerobic culture was performed, with negative results. Pain management was provided with meloxicam (0.2 mg/kg PO q24h) and tramadol (11 mg/kg PO q12h); the owner declined antimicrobial therapy. Three weeks later, radiographs revealed the progression of the lytic lesions to the distal femur, proximal tibia, and tarsal bones. One month later, the patient presented for left hindlimb swelling. Follow-up radiographs revealed a total loss of the right stifle and the tibiotarsal and tarsometatarsal joints, as well as new lytic lesions at the left stifle and tibiotarsal joints. Based on the grave prognosis, the lizard was euthanized. At necropsy, granuloma-like lesions were found at the stifles and the coxofemoral, tibiotarsal, and tarsometatarsal joints. Imprints made from these lesions revealed acid-fast bacilli. No other gross lesions were identified. Microscopic review of the tissues revealed severe granulomatous osteomyelitis and myositis, as well as a severe histiocytic and heterophilic hepatitis with acid-fast intrahistiocytic bacilli. Polymerase chain reaction results confirmed these bacteria as nontuberculous mycobacteria. To the authors' knowledge, this is the first postmortem report of septic arthritis associated with Mycobacterium spp. in a bearded dragon; only two other cases have been reported, both without postmortem findings.

Staphylococcal intramammary infection dynamics and the relationship with milk quality parameters in dairy goats over the dry period

The objectives of this study were (1) to report the rates of new intramammary infection (IMI) and spontaneous IMI cure over the dry period in 3 dairy goat herds; (2) to evaluate the factors predicting infection dynamics over the dry period; and (3) to define milk quality parameter thresholds that predict infection dynamics over the dry period. Two consecutive udder-half milk samples were collected 10 to 14 d apart before dry-off from 288 goats in 3 herds, and 2 consecutive udder-half samples were collected 7 to 14 d apart in the following lactation, with the first sample being collected ≤10 d in milk, from 200 of the same goats. In 2 of the herds, udder-half milk samples were also collected at the same time points (n = 312 halves; 157 goats) for measurement of milk quality parameters. Standard aerobic culture of milk samples was performed for the detection of mastitis pathogens. To rule out the presence of Mycoplasma spp. IMI, milk samples were also cultured on modified Hayflick medium. Non-Mycoplasma isolates were speciated using MALDI-TOF mass spectrometry. Staphylococcal isolates, when not identified by MALDI-TOF, were speciated using partial gene sequence analysis of rpoB or tuf. When >1 sample from an udder half yielded the same species, available isolates from the first and last positive samples for that species were strain-typed using pulsed-field gel electrophoresis. Incidence of new IMI and cure rate were computed. Generalized linear mixed regression models were built to evaluate the associations between new IMI and pre-dry somatic cell score (SCS), between IMI persistence and half-level SCS, and between IMI persistence and pre-dry IMI species. Thresholds for pre-dry SCS and lactose concentration were computed to predict IMI persistence. Overall, 12.6% (48/380) of halves had a persistent IMI. Cumulative incidence of new IMI over the dry period was 13.2%, and cure rate was 52.0%. Pre-dry SCS was not associated with odds of new IMI or IMI persistence. Pre-dry IMI species was not associated with odds of persistence. Lactose concentration was not associated with odds of persistence. Regardless of culture data, the optimal pre-dry SCS threshold to detect IMI that would persist into the next lactation was 8.7, with sensitivity and specificity of 50 and 73.8%, respectively. Further studies on the effect of control measures on species-specific incidence and cure rates during the dry period are warranted.

Case study: Evaluating quarter and composite milk sampling for detection of subclinical intramammary infections in dairy cattle

Our objective was to evaluate a 200,000 cells/mL somatic cell count (SCC) cut-point on both the quarter and composite level to determine its effectiveness at identifying subclinical mastitis infections in one commercial dairy herd in Central New York. Milk samples from 107 Holstein cows were used for analysis. All cows were eligible for enrollment provided they had 4 working udder quarters, were >14 and <365 d in milk, and had no clinical mastitis event or treatment with intramammary antibiotics ≤14 d prior to d of sampling. A total of 428 quarter and 107 composite samples from 34 primiparous and 73 multiparous animals were analyzed for total SCC and aerobic culture. Performance of SCC for identification of subclinically infected animals was evaluated against the gold standard aerobic culture. Sensitivities for a 200,000 cells/mL cut-point on both the quarter and composite basis were 28.6%, and specificities were 91.5% and 87.3% on the quarter and composite basis, respectively. Receiver operating characteristic (ROC) curves determined on a quarter basis found the cut-point that optimized the sensitivity and specificity of a positive culture was 32,000 cells/mL, with a sensitivity of 76.2%, a specificity of 62.4%, and an area under the curve (AUC) of 0.73. The ROC curve cut-point that optimized the sensitivity and specificity for the composite samples was 75,000 cells/mL, with a sensitivity of 57.1%, a specificity of 78.9%, and an AUC of 0.67. A large proportion of culture positive primiparous cows (38%) had low SCC (median of 101,000 cells/mL on the quarter and 80,000 cells/mL on the composite level), and therefore, when multiparous cows were examined separately, the cut-point that optimized sensitivity and specificity on the quarter basis increased to 645,000 cells/mL with a corresponding sensitivity of 34.8%, specificity of 97.5%, and AUC of 0.65. On the composite basis, the cut-point based on multiparous cows only was 152,000 cells/mL, with a corresponding sensitivity of 60.0%, and specificity of 82.0%, and an AUC of 0.65. Our data indicate that the 200,000 cells/mL cut-point was inefficient in identifying subclinically infected animals, regardless of whether quarter or composite sampling was used. The low prevalence of subclinical infections as well as the large proportion of minor pathogens, especially in primiparous animals, contributed to this inefficiency. This case study provides evidence that, with continued improvement upon mastitis control and reduction in major mastitis pathogens, blanket cut-points may no longer provide the same diagnostic usefulness as they once did.

Meningitis caused by Porphyromonas endodontalis detected by PCR amplification and sequencing of 16S rRNA genes direct from cerebrospinal fluid and cerebral tissue

Introduction: Porphyromonas endodontalis (formerly Bacteroides endontalis) is a black-pigmented, non-motile, obligate anaerobe. It has an established role in endodontal infections and has rarely been isolated from other sites. To the best of our knowledge, this is the first time that this organism has been detected as the sole cause of meningitis. This was made possible by the use of direct-on-sample 16S rRNA gene amplification and sequencing. Case presentation: We present a case of meningitis that progressed to a cerebral abscess in an elderly but otherwise immunocompetent male. Despite broad-spectrum antibiotic therapy, the patient died. Conclusion: Although anaerobic meningitis is rarely described, it should be considered if there are clinical and biochemical features of meningitis but no organism isolated on standard aerobic culture.

Aerobic bacterial flora of the normal conjunctiva at high altitude area of Shimla Hills in India: a hospital based study.

To study the normal aerobic conjunctival flora in lower to mid Himalayan region of Shimla Hills. Samples from normal conjunctiva of 200 individuals above the age of 13 years who visited Indira Gandhi Medical College Hospital Shimla for refraction or cataract surgery were taken by anaesthetising the conjunctival sac with sterile 4% Xylocaine solution. Samples were obtained by gently rubbing lower fornix with a sterile cotton wool swab moistened with normal saline and keeping the eye lids wide apart to avoid contamination from lid margins. It was immediately inoculated in Brain Heart infusion and subjected to standard aerobic culture and identification techniques. Totally, 72 eyes (36%) conjunctival sacs were sterile. Predominant aerobes isolated were Staphylococcus spp. in 120 (60%) followed by Haemophilus in 16 (8%), diphtheroids in 10 (5%), and Escherichia.coli (E. coli) in 4 (2%). A single aerobe was isolated from 98 eyes (49%) while 30 eyes (15%) yielded more than 1 aerobe. Staphylococcus epidermidis (S. epidermidis) was the most common bacterium, found alone in 58 eyes (29%) and in combination with another aerobe in 30 eyes (15%), followed by Staphylococcus aureus (S. aureus) in 18 (9%) and 10 eyes (5%) respectively. S. epidermidis is the most common commensal organism followed by Haemophilus species. diphtheroids occupied the third position which otherwise are found more abundantly in literature. Pathogens like S. aureus, Staphylococcus citreus and E. coli were also found. Therefore, preoperative administration of topical broad spectrum antibiotics is extremely important in prophylaxis against ocular infection. The variation in microflora of normal conjunctiva in this part of world can be attributed to geographical, climatic and ethnic characteristics of the population under study.