mBiosphere

Classic spotlights: penicillin for the masses

The Journal of Bacteriology is celebrating its centennial this year – one hundred years of valuable scientific discoveries! To illustrate the wide variety of noteworthy results published over the years, the journal is publishing a Classic Spotlight series, in which experts in the field highlight some of their favorite studies from past decades. Subjects covered so far include quorum sensing, fruiting bodies, biofilms, bacteriophage, and even the discovery of a second membrane around Gram-negative bacteria – all important core microbiology concepts. And we’re not even a quarter through 2016 yet!

In a new Editorial by Katrina Forest and Ann Stock, the discovery of penicillin is discussed – a notable milestone in our fight against infectious diseases. After its initial discovery by Alexander Fleming, the drug needed to be manufactured at a large enough scale before it could be distributed to sick individuals. Many scientific breakthroughs led to the ability to produce mass quantities, and the editorial highlights two: the discovery of naturally high volume-producing Penicillium notatum strains and the ability to purify penicillin from submerged cultures, rather than rely on surface-grown fungi.

The discovery of good penicillin-producing strains by Kenneth Raper, Dorothy Alexander, and Robert Coghill in 1944 involved tenacity and serendipity. They surveyed rotting fruit from produce markets in search of a more potent Penicillium species (hence the cover image, above left, of the current issue, illustrating a technician examining a cantaloupe). 241 strains were tested qualitatively for their ability to inhibit Staphylococcus growth, resulting in now-standard zones of inhibition (see figure, right). All the plugs were transferred and characterized by hand; as the Editorial indicates, this was high-throughput research in the 1940s!

Laboratory-scale production, described in 1945 by Andrew Moyer and Robert Coghill, addressed the problem of the time- and resource-expensive procedure of penicillin purification. This research used the strain described in the Raper study to test the effects of culture conditions on penicillin production. They found the optimal conditions for submerged cultures differed from those of surface cultures, and were then able to apply these conditions for industrial production of penicillin in tanks.

For more Classic Spotlights on seminal microbiology findings, stay tuned to mBiosphere and the Journal of Bacteriology!

-- Julie Wolf

Photo credits: Current JB Cover, Figure from Raper paper

Meta-analysis of urine pre-analytic practice reveals need for rigorous studies

Evidence-based medicine is the approach used by doctors to make rational clinical decisions based on rigorous, well-controlled studies. By minimizing hunches, gut feelings, and anecdotal evidence, physicians and patients can follow recommendations that are most likely to have a positive outcome. Practice of evidence-based medicine has improved patient quality of life, prevented unnecessary procedures, and improved patient recovery times.

Evidence-based medicine isn’t limited to patient therapy, however – it plays a strong role in diagnostics as well. Clinical microbiologists are using this approach to determine best-practices for sample collection and storage, two factors that can greatly impact the microbiological contents of a specimen. A recently published review in Clinical Microbiology Reviews investigated data supporting recommendations for urine collection and storage prior to processing. This is in an effort to standardize these practices among clinics for consistent and reproducible results.

Urine samples are some of the most frequently collected clinical specimens. Urinary tract infections (UTIs) are the most common bacterial infections the United States, accounting for 8.1 million physician visits annually, and urinanalysis is an important part of proper diagnosis. They are often caused by gram-negative bacteria, but ensuring culture of the causative microbe, and not a contaminant, is necessary to identify the correct etiology.

A number of guidelines exist regarding specimen collection, including those regarding washing before collection and clean catch of mid-stream urine. After collection, samples are either refrigerated or preserved with boric acid for up to 24 hours before processing. The panel of experts involved in the study delved into the literature to see how strongly the data supported these recommendations. This work was sponsored by the American Society for Microbiology in collaboration with the Center for Disease Control and Prevention's Division of Laboratory Programs, Standards, and Services through a Laboratory Medicine Best Practices Program Memorandum of Understanding.

The researchers focused on eight specific clinical questions (see inset, right). Beginning with over 5000 studies, they strategically concentrated the relevant articles to 171 using applicable search terminology. Exclusion of poor-quality data further winnowed the relevant articles down to a final 35 that met all criteria: 10 studies on storage and 25 on collection (14 studies on collection from children/infants, 8 studies with women subjects, and only 3 studies with men).

All the studies on specimen storage supported the use of either refrigeration or boric acid addition for sample preservation. The review panel further concluded that samples stored at room temperature for over four hours should be discarded, since overgrowth quickly occurs in this condition.

Sample collection literature was not as coinciding. While midstream clean-catch was associated strongly with reduced contamination in adults, the overall effect of perineal cleansing was uncertain for women. However, the overall strength of the body of evidence around these conclusions was low. Although there were slightly more studies involving children and infants, the results still varied. In this patient population, cleansing did reduce contamination for midstream collection. Diaper or sterile urine bag collection is often used for very young (read: not potty-trained) children, but these methods were not recommended by the panel.

The most important finding of this study may be the lack of rigorous studies examining urine collection and storage. Most studies were observational and poorly controlled; some failed to report data for all study participants. These are investigational aspects that can be more systematically addressed in future research. Better pre-processing specimen handling will decrease sample contamination events, increase the speed and accuracy of diagnoses, and lead to more consistent results. In the end, both patient and health-care provider benefit from more timely treatment and more efficient (and thus less expensive) health care costs.

These meta-analyses serve an additional purpose. Clinicians have many tasks pulling at their attention – reading and synthesizing all the relevant studies is something for which many simply don’t have time. Recommendations by those willing to dig deep into the literature provide a valuable resource for the entire field. The discovered gap in knowledge can guide future studies to develop these type of recommendations. To this end, the study included suggested questions and recommendations to help improve forthcoming project design and analysis.

-- Julie Wolf

Photo credits: Urinalysis video, Questions posed by CMR article, Medical Laboratory Scientist

MERS Research Yields Surprising Finding

In 2015, an unexpectedly large outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in South Korea had a disastrous impact on the whole country and highlighted scientists limited knowledge of this virus. Now, new research into this outbreak has revealed some very surprising findings.

MERS-CoV was first identified in Saudi Arabia in 2012. The virus has been mainly seen in Middle Eastern countries, with cases reported in at least 10 other countries in Europe, Asia, and the United States. The virus causes severe respiratory infection and has a worldwide mortality rate of approximately 35%. Similar to other coronaviruses, MERS-CoV utilizes a large surface spike glycoprotein to enter human CD26 cells and cause infection.

From May to July 2015, a large outbreak of MERS initiated by an infected traveler from the Arabian peninsula swept South Korea. In its wake, it left 186 confirmed cases and 38 deaths. “The unexpected outbreak raised strong concerns about the possible generation of mutant viruses and prompted us to investigate the MERS viruses infecting Korean patients,” said Nam Hyuk Cho, PhD, faculty member at the Seoul National University College of Medicine in Korea, and principal investigator of a new study reported this week in mBio, an online open-access journal of the American Society for Microbiology.

In the study, investigators isolated 13 new viral genomes from 14 infected patients with MERS treated during the outbreak. They found that 12 of the genomes had two specific point mutations (I529T and D510G mutations) in the receptor-binding domain (RBD) of the viral spike protein. Further analysis showed that the acquired mutations made the virus less virulent rather than more virulent.

“Strikingly, both mutations resulted in reduced affinity of RBD to human CD26 compared to wild-type RBD,” explained Dr. Cho. “This is an interesting strategy of coronavirus evolution to survive in nature and live together with the new host. The virus may tune down its power to attack for the sake of longer survival in the new host.”

Dr. Cho pointed out that the kinds of changes seen in the viral spike protein have been reported previously in the study of severe acute respiratory syndrome. (SARS)-CoV, another animal-derived coronavirus that spread worldwide in 2002 and 2003. At that time, scientists said changes in the spike protein toward reduced affinity to the human CD26 receptor might be driven by immunological pressure, such as neutralizing antibodies. Neutralizing antibodies defend a cell from an antigen or infectious body by neutralizing any effect it has biologically. “This type of viral evolution during host changes, camel-to-human and human-to-human spread, may occur for host adaptation and immune escape,” said Dr. Cho. “The mutations may impair viral fitness and virulence to human, but provide more of a chance to escape the antiviral immunity.”

The findings have implications for the development of a vaccine for MERS. Currently, most vaccine trials for MERS prevention are using the spike antigen to generate effective neutralizing antibodies against it. “Strategies for vaccine development also need to consider the chance of emergence of neutralizing antibody-escape mutants,” said Dr. Cho. “Vaccines for MERS need to target the more stable and conserved region of the spike.”

 

-- Kate O'Rourke