Bacteria have always been promiscuous: Different resistant E coli strains can cross-protect

Two strains of bacteria resistant to different antibiotics can protect each other in an environment where both drugs are present, according to the first experimental study of microbial cross-protection published last week in Proceedings of the National Academy of Sciences (PNAS).

bob-carol-ted-aliceResearchers from the Massachusetts Institute of Technology (MIT) Department of Physics explored the potential of mutualism—an interaction that benefits two different species—on two strains of Escherichia coli, one of which was resistant to ampicillin and the other resistant to chloramphenicol.

Though the type of mutualism known as cross-protection, in which species depend on each other for survival in a challenging environment, has been observed in larger animals, it has not previously been observed experimentally in bacterial populations, the authors noted.

Cross-protection in drug-resistant E coli depended on a host of factors, including characteristics of each resistant strain, presence and amount of antibiotics, dilution and oscillation of bacterial population abundance, and invasion by other bacteria.

The role of enzyme deactivation

Strains of E coli express antibiotic resistance by producing defensive enzymes that destroy the drug. Resistant strains can often protect drug-susceptible pathogens through enzyme deactivation if they are able to quickly remove the antimicrobials from the environment, the authors said.

During a 10-day experiment, researchers exposed an ampicillin-resistant and a chloramphenicol-resistant E coli strain to mixed concentrations of the antibiotics that should have killed each strain alone. Ampicillin-resistant E coli cannot survive alone in a chloramphenicol concentration above 2.2 micrograms per milliliter (mcg/mL), and a chloramphenicol-resistant strain will be destroyed when exposed to 2 mcg/mL of ampicillin.

Even when bacterial populations were diluted each day by transferring 1% of the colonies to a new test tube containing antibiotics, the strains were able to protect each other against drug concentrations fourfold higher than amounts lethal to one strain alone.

“A coculture of the two strains can survive above the concentrations at which the individual strains survive alone, indicating that the two populations form an obligatory mutualism,” the authors wrote.

e_coli-eraxion-istockA key contributor to cross-protection between two resistant bacterial strains was each population’s ability to oscillate in size when diluted daily in a solution containing ampicillin and chloramphenicol. Oscillation cycles lasted for 3 days and involved massive changes in the percentage of each strain while the total size of the bacterial population remained stable.

During a 3-day oscillation cycle, the ampicillin-resistant strain grew in abundance as it deactivated the antibiotic. This activity allowed the chloramphenicol-resistant strain to grow, removing chloramphenicol from the environment, until the ampicillin-resistant strain could increase again, thus continuing a cycle in which the strains protected and overtook each other in abundance. Populations varied by up to 1,000% over the 3-day range, the authors said.

Oscillations in abundance occurred because of the daily dilution in an antibiotic-rich environment and were not related to different natural growth rates in each strain, the author said. The cross-protective effect achieved by the oscillations appeared stable, with shifts in each strain’s growth and relative proportion likely being sustainable over time.

“Because these oscillations occurred with a period (3 days) longer than the period of the daily dilution (1 day), they were not a trivial consequence of the daily growth-dilution cycle,” the authors said.

Oscillation cycles had to be precisely balanced to prevent the cross-protective interaction’s total collapse. At chloramphenicol concentrations of 7.6 mcg/mL, both bacterial populations were able to achieve stable oscillations and coexist. When exposed to chloramphenicol levels of 17.1 mcg/mL, however, the oscillations became erratic, the sustainable 3-day cycle was lost, and the interaction collapsed, the probability of which increased at chloramphenicol concentrations of 38.4 mcg/mL.

Frequently diluting small concentrations of bacteria in media containing 10 mcg/mL of ampicillin and 5.1 mcg/mL of chloramphenicol allowed the bacterial populations to form a stable cross-protective relationship.

“In a continuous culture experiment in which the antibiotics are continuously added (and cells continuously removed), there will be no oscillations in the population abundances, and instead the ratio between the two strains should approach a stable equilibrium,” the authors wrote.

The introduction of an individual E coli strain resistant to both antibiotics also caused the cross-protective behavior to collapse, the authors said.

When researchers added a small number of double-resistant E coli cells to the dilutions at the beginning of the seventh growth cycle, the double-resistant strain displaced the ampicillin-resistant E coli and co-existed with the chlorampenicol-resistant strain in a solution containing concentrations of ampicillin at 10 mcg/mL and chloramphenicol at 7.5 mcg/mL.

“A double-resistant strain can invade the mutualism and cause the oscillations to vanish, illustrating that the existence of oscillations depends on how resistance is allocated in the microbial population,” the authors said.

In the absence of a multi–drug-resistant microbial invasion, horizontal gene transfer in the cross-protective co-culture could create a double-resistant mutant strain, the authors said. In addition, “the cooperative nature of antibiotic deactivation could allow a sensitive strain to use the two mutualists for protection.”

Implications for infection and resistance

Because cross-protection often arises as a strategy to help survive a harsh environment, greater understanding is needed about both the environment and the microbial population dynamics, including the role of oscillations in strain abundance, under which mutualistic behavior allows bacterial resilience in the presence of antibiotics. The authors note that little knowledge is available about the role and cause of oscillations in stabilizing population abundance even as they occur in more easily observable organisms, such as the Canada lynx and snowshoe hare.

Many cross-protective relationships are the result of two organisms co-evolving in the same environment, though this is not the case with E coli strains. Mutualistic behavior in this case is thought to arise as a result of exposure to antibiotics, the authors said.

“Whether an interaction is cooperative or competitive can depend on the environment,” the authors wrote, adding that exposing cross-protective strains to antibiotics can fuel the development of multi-drug resistance by buying time for further evolutionary adaptation.

Nickelback? Really? So much for Trudeau being cool and Shiga-toxin producing E. coli in pigs

Still a deafening silence from public health types over whether or not people are definitively sick or just sick from Cantran Meat Co. raw pork and pork organ products linked to an E, coli O157 outbreak in Alberta.

TrudeauNickelbackSmallIt is a long weekend in Canada – Queen Victoria’s birthday or something as an excuse to go camping in the cold and mark the start of summer – so don’t expect anything public soon.

But an astute conversationalist did send along this abstract from last month to help answer the question, what is Shiga-toxin producing E. coli doing in pig?

Abstract

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-.

STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), andstx1c (1%).

kid_pig_kissThe STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2aand stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes,hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB.

The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

Frontiers in Microbiology, 21 April 2016, http://dx.doi.org/10.3389/fmicb.2016.00574

Gian Marco Baranzoni, Pina M. Fratamico, Jayanthi Gangiredla, Isha Patel, Lori K. Bagi, Sabine Delannoy, Patrick Fach, Federica Boccia, Aniello Anastasio and Tiziana Pepe

http://journal.frontiersin.org/article/10.3389/fmicb.2016.00574/full

Orwellian: People are sick, but CFIA plays word games with E coli O157 notice

As usual, the Canadian Food Inspection Agency buries the lede at the bottom:

george-orwell-6“There have been no illnesses definitively linked to the consumption of these products.”

Yet the recall of Cantran Meat Co. raw pork and pork organ products from the marketplace due to possible E. coli O157:H7 contamination was triggered by findings of CFIA, Alberta Health Services, and Alberta Agriculture and Forestry during the investigation into a foodborne illness outbreak in Alberta.

Way to mention government agencies and snivel servants, way not to mention sick people.

The affected raw pork and pork organ products, supplied by Cantran Meat Co. Ltd., may have been transformed into raw muscle meat cuts, ground pork, sausages, and raw ready-to-eat products. The products, which have been sold fresh, have only been distributed in Alberta.

The affected products are known to have been sold or distributed by the companies from April 28, 2016 up to and including May 14, 2016. The products may have been sold pre-packaged or clerk-served, with or without a label. Consumers who are unsure if they have the affected products are advised to check with their retailer.

News amplification: Mad cow disease far greater impact on beef purchases than E. coli

In December 2003, Bovine Spongiform Encephalopathy (BSE) was discovered in the United States. This food safety event received extensive media coverage and prompted changes in regulatory controls.

bse.cow.may.16Using a panel selection model, we show that prior to December 2003, ground beef recalls had no impact on household purchases of ground beef, even for households that were located in the recall-defined geographic areas. However, we find robust evidence that the 2003 BSE event caused a change in the way people view and respond to recalls of ground beef, a change that persisted for at least two years following the BSE event.

The average impact of a ground beef recall in the post-BSE period is a 0.26 lb per person reduction in retail purchases of ground beef. A decline in purchases of this magnitude would result in over $97 million in losses to the beef industry in a two-week period following a nationwide recall.

This dwarfs the economic impacts of directly removing recalled beef from supply chains and provides FSIS increased regulatory power due to higher overall industry costs associated with food safety violations.

Changes in U.S. consumer response to food safety recalls in shadow of a BSE scare

Mykel Taylora, H. Allen Klaiberb, Fred Kuchlerc

Food Policy, Volume 62, July 2016, Pages 56-64, doi:10.1016/j.foodpol.2016.04.005

http://www.sciencedirect.com/science/article/pii/S0306919216300239

Penn. schools get grant to study shiga-toxin producing E. coli

The Souderton Area School District received $250,000 from the U.S. Department of Agriculture on Monday to help fund a career-exploration program for students who want safe food.

stec.cap.SoudertonThe money is part of a $25 million grant named after a bacteria commonly associated with foodborne illnesses.

The STEC-CAP grant, also known as the Shiga toxin Escherichia coli Coordinated Agricultural Project, will connect Souderton students to researchers across the country whose objectives are to identify and eliminate pathogenic E. coli on food, said Kyle D. Longacre, the high school’s assistant principal.

The University of Nebraska-Lincoln directs the research, which involves participants from 13 colleges and universities.

According to Longacre, a partnership between the USDA and Souderton schools has prospered since 2014 when 10 high school teachers were trained about food safety methods, bacterial growth labs and the latest research related to Shiga toxin E. coli.

The money will also help Souderton’s Pathway 360 program, which places motivated students in the professional workplace with mentors.

Longacre, who leads Pathway 360, said the high school would use the money to spread the word about food safety and to get students to consider a career in food science and agriculture.

“It’s been a tremendously creative program,” said Dr. Rodney Moxley, a professor in the School of Veterinary Medicine and Biomedical Science at the University of Nebraska-Lincoln. “Pathway 360 has its own inherent accomplishments in that you are helping students to get jobs.”

According to the district, the money also will help fund the possible expansion of partnerships with Upper Perkiomen, Norristown and other school districts.

Longacre said students in Pathway 360 take part in an initiative called “Message to the Masses” in which Souderton’s Design, Marketing and Communications club partnered with three advertising agencies to create slogans for the USDA’s program on safe handling of beef.

Students in the design and marketing club presented their ideas to advisers following Monday’s announcement of the grant. The ideas were then critiqued by advisers with the STEC-CAP grant program. Longacre said the advisers will select the best idea, and the USDA will fly five or more Souderton students to Nebraska in June to present their ideas to a national council of STEC-CAP advisers.

Throwing stones from Haaarvaaard: The cost of a sick customer

Harvard picked an easy target, offering its management insight to Chipotle, but I’ve yet to see a paper about how the venerable Haaarvaaard Faculty Club managed to sicken patrons not once, but twice with Norovirus in 2010.

simpsons.harvardAccording to PR from Haaarvaaard, Chipotle has seen its shares tumble and recently reported its first-ever quarterly loss after the incident, which began in October when more than 50 people in 11 states were sickened by an initial E. coli outbreak.

The chain restaurant, which uses the tagline “Food with Integrity,” has prided itself on avoiding artificial ingredients, opting instead to use a relatively short supply chain of local growers for many of its ingredients.

That strategy just might have been part of its problem, says John A. Quelch, the Charles Edward Wilson Professor of Business Administration at Harvard Business School and Professor in Health Policy and Management at the Harvard T.H. Chan School of Public Health.

Quelch, who teaches a course to Harvard business and public health students called Consumers, Corporations and Public Health, says food safety is more challenging than ever for three reasons:

  • the globalization of the food business;
  • global food safety standards are lacking; and,
  • food safety problems can be quite costly.

Thanks for the insight. Back to hockey.

Fresh or frozen, deep-fried or baked: Reducing E. coli in meatballs

I’d always use a tip-sensitive digital thermometer to account for variations in cooking appliances, degrees of thawdiness (yes, I’m making up words) initial bacterial loads.

meatballs2We investigated the effects of deep-frying or oven cooking on inactivation of Shiga toxin–producing cells of Escherichia coli (STEC) in meatballs.

Finely ground veal and/or a finely ground beef-pork-veal mixture were inoculated (ca. 6.5 log CFU/g) with an eight-strain, genetically marked cocktail of rifampin-resistant STEC strains (STEC-8; O111:H, O45:H2, O103:H2, O104:H4, O121:H19, O145:NM, O26:H11, and O157:H7). Inoculated meat was mixed with liquid whole eggs and seasoned bread crumbs, shaped by hand into 40-g balls, and stored at −20°C (i.e., frozen) or at 4°C (i.e., fresh) for up to 18 h. Meatballs were deep-fried (canola oil) or baked (convection oven) for up to 9 or 20 min at 176.7°C (350°F), respectively. Cooked and uncooked samples were homogenized and plated onto sorbitol MacConkey agar with rifampin (100 μg/ml) followed by incubation of plates at 37°C for ca. 24 h. Up to four trials and three replications for each treatment for each trial were conducted.

Deep-frying fresh meatballs for up to 5.5 min or frozen meatballs for up to 9.0 min resulted in reductions of STEC-8 ranging from ca. 0.7 to ≥6.1 log CFU/g. Likewise, reductions of ca. 0.7 to ≥6.1 log CFU/g were observed for frozen and fresh meatballs that were oven cooked for 7.5 to 20 min.

This work provides new information on the effect of prior storage temperature (refrigerated or frozen), as well as subsequent cooking via deep-frying or baking, on inactivation of STEC-8 in meatballs prepared with beef, pork, and/or veal. These results will help establish guidelines and best practices for cooking raw meatballs at both food service establishments and in the home.

Effect of deep-frying or conventional oven cooking on thermal inactivation of Shiga toxin–producing cells of Escherichia coli in meatballs

Journal of Food Protection®, Number 5, May 2016, pp. 696-889, pp. 723-731(9)

Porto-Fett, Anna C. S.; Oliver, Michelle; Daniel, Marciauna; Shoyer, Bradley A.; Stahler, Laura J.; Shane, Laura E.; Kassama, Lamin S.; Jackson-Davis, Armitra; Luchansky, John B.

http://www.ingentaconnect.com/contentone/iafp/jfp/2016/00000079/00000005/art00005

 

Pathogens? In NZ milk? Never …

Zoonotic bacteria such as Campylobacter, Listeria, and Shiga toxin–producing Escherichia coli have been found in bulk tank milk in many countries, and the consumption of raw milk has been implicated in outbreaks of disease in New Zealand.

milk.pathogens.nzFecal contamination at milking is probably the most common source of pathogenic bacteria in bulk tank milk.

Raw milk was collected from 80 New Zealand dairy farms during 2011 and 2012 and tested periodically for Campylobacter, E. coli O157, Listeria monocytogenes, and Salmonella. Milk quality data such as coliform counts, total bacterial counts, and somatic cell counts also were collected. By treating the total bacterial count as a proxy for fecal contamination of milk and utilizing farm and animal level prevalence and shedding rates of each pathogen, a predictive model for the level of pathogenic bacteria in bulk tank raw milk was developed. The model utilizes a mixture distribution to combine the low level of contamination inherent in the milking process with isolated contamination events associated with significantly higher pathogen levels. By simulating the sampling and testing process, the predictive model was validated against the observed prevalence of each pathogen in the survey.

The predicted prevalence was similar to the observed prevalence for E. coli O157 and Salmonella, although the predicted prevalence was higher than that observed in samples tested for Campylobacter.

Estimating bacterial pathogen levels in New Zealand bulk tank milk

Journal of Food Protection®, Number 5, May 2016, pp. 696-889, pp. 771-780(10)

Marshall, J. C.; Soboleva, T. K.; Jamieson, P.; French, N. P.

http://www.ingentaconnect.com/contentone/iafp/jfp/2016/00000079/00000005/art00011

Try harder: UK petting farm ‘doing all it can’ after E. coli outbreak

The owners of a petting farm at the centre of a parasitic disease outbreak that has left dozens ill said they are working with the local authority to investigate its cause.

swithern.farmIan and Angela Broadhead, who run Swithens Farm, in Rothwell, Leeds, have reassured visitors that their “health, safety and welfare” is of “utmost importance” to them as they continue to work with public health experts.

The petting farm has been linked to 29 cases of cryptosporidiosis, and two cases of E.coli O157.

The Broadhead family said: “As a small family-run business the health, safety and welfare of our visitors is of utmost importance to us all.

handwash.UK_.petting.zoo_.09Between January and May 2015 around 130 people were affected by outbreaks of cryptosporidiosis linked to petting farms in England.

PHE has advised all visitors to wash their hands after touching animals.

Handwashing, however, is never enough.

A table of petting zoo outbreaks is available at http://barfblog.com/wp-content/uploads/2016/05/Petting-Zoo-Outbreaks-Table-5-5-16.xlsx

Best practices for planning events encouraging human-animal interations

Zoonoses and Public Health 62:90-99

Erdozain , K. KuKanich , B. Chapman and  D. Powell, 2015

http://onlinelibrary.wiley.com/doi/10.1111/zph.12117/abstract?deniedAccess

Educational events encouraging human–animal interaction include the risk of zoonotic disease transmission. ‘It is estimated that 14% of all disease in the USA caused by Campylobacter spp., Cryptosporidium spp., Shiga toxin-producing Escherichia coli (STEC) O157, non-O157 STECs, Listeria monocytogenes, nontyphoidal Salmonella enterica and Yersinia enterocolitica were attributable to animal contact. This article reviews best practices for organizing events where human–animal interactions are encouraged, with the objective of lowering the risk of zoonotic disease transmission.

petting1-791x1024

petting2-791x1024 

Lettuce rinse with cheese whey can reduce pathogens

Cheese whey fermented by an industrial starter consortium of lactic acid bacteria was evaluated for its antibacterial capacity to control a selection of pathogenic bacteria. For their relevance on outbreak reports related to vegetable consumption, this selection included Listeria monocytogenes, serotype 4b, Escherichia coli O157:H7, and Salmonella Goldcoast.

cheese.wheyOrganically grown lettuce was inoculated with an inoculum level of ∼107 colony-forming unit (CFU)/mL and was left for about 1 h in a safety cabinet before washing with a perceptual solution of 75:25 (v/v) fermented whey in water, for 1 and 10 min. Cells of pathogens recovered were then counted and their number compared with that obtained for a similar treatment, but using a chlorine solution at 110 ppm.

Results show that both treatments, either with chlorine or fermented whey, were able to significantly reduce (p < 0.05) the number of bacteria, in a range of 1.15–2.00 and 1.59–2.34 CFU/g, respectively, regarding the bacteria tested. Results suggest that the use of fermented whey may be as effective as the solution of chlorine used in industrial processes in reducing the pathogens under study (best efficacy shown for Salmonella), with the advantage of avoiding health risks arising from the formation of carcinogenic toxic chlorine derivates.

Preliminary study on the effect of fermented cheese whey on Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Goldcoast populations inoculated onto fresh organic lettuce

Maria I.S. Santos,1,2,3,4 Ana I. Lima,4 Sara A.V.S. Monteiro,4 Ricardo M.S.B. Ferreira,4 Laurentina Pedroso,3 Isabel Sousa,2 and Maria A.S.S. Ferreira1

1Microbiology Laboratory, Department of Natural Resources, Environment and Territory, DRAT, LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal.

2Eco-Processing of Food and Feed, CEE, LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal.

3Faculty of Veterinary Medicine, Universidade Lusofona de Humanidades e Tecnologias, Lisbon, Portugal.

4Disease & Stress Biology, DRAT, LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal.

Foodborne Pathogens and Disease, doi:10.1089/fpd.2015.2079

http://online.liebertpub.com/doi/abs/10.1089/fpd.2015.2079