Case Report: The Endemic Spread of Escherichia coli Positive for Cytotoxic Necrotizing Factor Type 1 in a Canine Research Facility and Impact of Infection in Neonatal Puppies

Introduction

Bacterial infection is one of the most common causes of neonatal morbidity and mortality in puppies, second only to respiratory distress syndrome.¹ Among the various causative bacteria, Escherichia coli is the most common isolate.^1,2 Outbreaks within a dog colony, or even a single litter, require rapid diagnosis and treatment to avoid neonatal mortality. Escherichia coli is a highly diverse group of bacteria including nonpathogenic types and numerous pathotypes, each with unique virulence factors and pathogenic properties. Spread via fecal matter exposure, among other modes of transmission, nonpathogenic and pathogenic types (pathotypes) of E. coli can colonize outside of the intestinal tract, leading to infection when left untreated.³ In humans, E. coli pathotypes have been linked to neonatal meningitis, urinary tract infections, septicemia, and respiratory infections such as pneumonia.⁴ Furthermore, infection with E. coli pathotypes has been related to development of necrotizing pneumonia.⁵ Various studies of these illnesses and the bacteria have associated disease progression with the cytotoxic necrotizing factor 1 (CNF1) protein, encoded by the virulence gene cnf1.^5–7 In general, CNF1 leads to malfunction of Rho signaling in host cells,⁸ which plays a key role in cell cycle, membrane trafficking, microtubule stability, and many other pathways in the body.⁹ Therefore, CNF1 has a significant impact on the regulation of cell survival.

This case report demonstrates the relationship between cnf1⁺ E. coli and disease progression resulting in neonatal canine infection and death. In September 2013, a canine research colony at Michigan State University’s (MSU) canine research vivarium began experiencing an unexplained increase in puppy mortality. Researchers observed rapid deterioration of puppies whelped to dams that ultimately tested positive for cnf1⁺ E. coli. Fecal and vaginal samples were taken from adult canines to identify carriers of the bacterial strain. Adult carriers remained healthy, with the most significant impact occurring in newborn puppies. Clinical signs in neonates included weight loss, respiratory distress, lethargy, and other indicators of infection. An analysis of 2 litters, totaling 14 puppies with 13 fatalities and 1 survivor, revealed that both dams had tested positive for cnf1 at the time of whelping. Further testing revealed other affected breeding females within the vivarium, which were subsequently treated prior to whelping and did not experience neonatal mortality. The following data showcase the effects of cnf1⁺ E. coli infection on both canine adults and neonates and explore the common presenting signs observed in neonatal puppies infected by cnf1⁺ E. coli, a topic in need of further research to fully understand its impact.

Case Report

Materials and Methods

Deceased puppies were submitted to the MSU VDL (Lansing, MI) for necropsy and culture of tissue samples. Biologic samples were collected from adult male and female canines. Vaginal and rectal samples were collected from female breeder canines by inserting a sterile Culturette several inches into the vagina and rolled from side to side. A concurrent sample was taken from the rectum in a similar manner. Preputial samples were collected from male canines by inserting a sterile Culturette (Becton Dickinson, Franklin Lakes, NJ) several inches into the prepuce and rolled from side to side. Samples were taken the same day to the MSU VDL, where processing began immediately.

Surfaces of the whelping box and whelping room floor were sampled using sterile cotton swabs (BD ESwab; Becton Dickinson, Franklin Lakes, NJ) that were premoistened with the transport buffer. The swab was rolled over multiple areas on each surface of interest and placed back into the transport tube. Samples were taken the same day to the MSU VDL, where processing began immediately.

Culturettes and swabs were plated to tryptic soy agar with 5% sheep blood (Remel R01202; Thermo Fisher Scientific, Waltham, MA), and to Columbia CNA (colistin + nalidixic acid) which is a selective medium for Gram-positive bacteria. It is also used to demonstrate hemolytic reactions. This medium is used for the analysis of mixed flora specimens, such as the genital tract, agar with sheep blood, and MacConkey agar (Remel R01552; Thermo Fisher Scientific, Waltham, MA). The plates were streaked to isolate colonies and were incubated for 3 days at 35 to 37 °C in 5% to 8% CO₂. Bacterial colonies were selected and confirmed using a MALDI-TOF MS Microflex LT Biotyper (Bruker Daltonics, Bremen, Germany), according to the manufacturer’s protocol. PCR was performed on any identified E. coli isolates for the presence of the virulence gene cnf1. In brief DNA was extracted from the isolated bacteria and PCR was performed using the primers CNF1 forward (5′-TATTCTGCAGTGACCGGATCTC-3′) and CNF1 reverse (5′-TATCCCCAAAGCTTCCAGAACC-3′). The PCR parameters were 95 °C for 5 minutes, followed by 32 cycles of denaturation at 95 °C, annealing at 50 °C for 45 seconds, and extension at 72 °C for 45 seconds. The PCR product was resolved on a 1.5% agarose gel. The CNF1 PCR was validated using the uropathogenic J96 E. coli strain, and the PCR amplicon (145 bp) was Sanger sequenced for confirmation.

Antimicrobial susceptibility testing was performed (Sensititre COMPGN1F panel; Thermo Fisher Scientific, Waltham, MA) for isolated E. coli to determine the minimum inhibitory concentration. Susceptibility was determined for each antibiotic using the Clinical and Laboratory Standards Institute performance standards for antimicrobial susceptibility testing. The standards (breakpoints) used were those for E. coli in canines except for tetracycline/doxycycline (equine break points), while human breakpoints were used for imipenem and chloramphenicol.

Antimicrobial sensitivity among cnf1⁺ E. coli between September 2013 and December 2020 was summarized, and change over time was assessed using the Cochran-Armitage test for trend (SAS 9.4). For the purposes of data presentation, intermediate minimum inhibitory concentration values were considered resistant. For ease, data for the β-lactams (cefazolin, cefovecin, cefpodoxime, and ticarcillin/clavulanate) are shown in one graph, and the data for chloramphenicol, doxycycline, and trimethoprim sulfamethoxazole are shown in another graph.

Results

A total of 10 puppies were submitted for necropsy from the 2 affected litters; all 10 had full postmortem examinations and major tissues were examined microscopically for 8 puppies (Table 2). The other 3 puppies that died (Table 1, puppies D-F) were not submitted for necropsy. The deceased puppies were in fair (weights of 190.2 to 210.9 g) to good (weights of 205.1 to 305.5 g) body condition, <1 week old, and with mild postmortem autolysis. Gross findings included pulmonary congestion, subcutaneous edema, hepatocellular necrosis, and fibrin on the capsular surface of the liver.

PCR for canine herpesvirus was negative. Bacterial culture on each lung sample from puppies A, B, and C, as well as pooled lung samples from puppies X1-X5, showed numerous β-hemolytic Streptococcus group G, few Staphylococcus pseudintermedius, and numerous E. coli. Pooled liver samples from puppies A, B, and C and puppies X1-X5 showed numerous β-hemolytic Streptococcus group G, moderate S. pseudintermedius, and moderate E. coli. The E. coli found in both the lung and liver samples tested positive for cnf1 virulence factor. Sections of brain, lung, heart, thymus, spleen, liver, kidney, trachea, esophagus, pancreas, lymph node, stomach, and intestine were examined from 8 of the deceased puppies. Figure 1 shows the range of lesions in puppies infected with cnf1⁺ E. coli infection. Major histopathologic features noted in these puppies include hemorrhage, necrosis, and less frequently neutrophilic infiltration in the alveoli of the lung and liver parenchyma. Intravascular and extravascular bacteria were noted in the heart, liver, lung, and blood vessels throughout the body. Puppies in the first litter all had lesions of necrotizing pneumonia, whereas puppies in the second litter all had some degree of hepatocellular necrosis and suppurative embolic pneumonia. One puppy in the second litter contained some vascular thrombi with embedded bacteria and vasculitis (Figure 1C).

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Following the puppy mortality, biologic and environmental sampling was conducted. Vaginal and rectal swabs from 5 female breeder canines were analyzed for the presence of cnf1⁺ E. coli. This group included the mothers of the 2 litters that had become ill and 3 pregnant females. In addition, preputial culture swabs were analyzed from 2 of the colony males who were known to have bred with carrier females on multiple occasions to assess possible carrier status in these breeding males. The results of the swabs identified the presence of cnf1⁺ E. coli in all female canines tested (Table 3). The mothers of the 2 litters with increased mortality were each positive from either the vaginal or rectal swab. One additional pregnant dog was positive from both swabs, and the remaining 2 pregnant dogs were only positive from vaginal swabs. Preputial samples collected from the 2 males used for breeding in affected litters tested negative.

The swabs from the whelping boxes and whelping room floor were positive for S. pseudintermedius, Staphylococcus hominis, nonhemolytic Streptococcus sp., Candida parapsilosis, Bacillus spp., Aspergillus spp., and unspecified fungus. However, samples tested negative for E. coli.

Between September 2013 and December 2020, 112 culture swabs were collected from all breeding females within the vivarium. Of these, 47.3% (53/112) tested positive for cnf1⁺ E. coli. Figure 2 shows that the frequency of dogs testing positive ranged from 17.6% (2019) to 68.8% (2017). Although no further neonatal death resulted from these bacteria following the implementation of uniform testing of pregnant females at 4 weeks gestation, a proportion of breeding females continued to test positive each year.

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Of the 53 positive samples collected and tested between 2013 and 2020, antimicrobial susceptibility data were available for 81% (43 of 53 samples). Multiple cnf1⁺ E. coli colonies with unique susceptibility patterns were identified from 5 samples, providing a total of 49 isolates. All isolates identified over this period remained sensitive to amikacin, enrofloxacin, imipenem, marbofloxacin, piperacillin/tazobactam, orbifloxacin, and gentamicin, with the exception of one orbifloxacin-resistant and one gentamicin-resistant isolates.

Conversely, all isolates were resistant to cephalexin, amoxicillin/clavulanate, ampicillin, rifampin, penicillin, and tetracycline, with the exception that one isolate was sensitive to tetracycline. The remaining antibiotics produced varied sensitivity results (Figure 3). Isolates of cnf1⁺ E. coli displayed significantly increasing resistance to cefazolin, cefovecin, cefpodoxime, chloramphenicol, and doxycycline over time (P < 0.0001).

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Discussion

This case report summarizes the clinical presentation, gross and histopathologic findings, and changes to medical management protocols from the high puppy mortality caused by cnf1⁺ E. coli infection. While cnf1⁺ E. coli was isolated from all puppies and pregnant canines at the time, all samples from breeding males and the whelping environment were negative. In response to the increased puppy mortality in 2013, the MSU vivarium implemented a testing protocol for all pregnant canines; however, there was no uniform treatment protocol based on these results, which ultimately produced isolates with increasing resistance during the next 7 years.

Puppy mortality due to cnf1⁺ E. coli has been reported before.^7,10,11 A study analyzing the impact of cnf1 in mammalian cells found that this virulence factor resulted in tissue necrosis, cell multinucleation, apoptosis, and inhibition of cell repair mechanisms due to its impact on Rho signaling.¹⁰ The presence of cnf1⁺ E. coli was detected in the lung and liver collected from submitted puppies, and tissue necrosis was noted where advanced infection had taken hold. Many signs of disease, histopathologic lesions, and areas of bacteria localization were found to be the same between littermates. Areas of suppurative inflammation and necrosis were noted within the lungs of 4 puppies and the livers of 3 puppies. Intravascular bacteria were noted in the lungs of 2 puppies. Septicemia and pneumonia have been directly linked to cnf1⁺ E. coli infection in canines following colonization of the lung tissue.¹² Importantly, note that pneumonia can also result from the presence of Streptococci group G, which was isolated in the lung samples from both litters. However, the presence of such bacteria was most likely a result of weakened immune health due to the advanced E. coli infection, leaving affected puppies more susceptible to opportunistic bacteria.¹³ Furthermore, only cnf1⁺ E. coli was consistently isolated in tissue cultures for all afflicted puppies. In general, the final cause of death was identified as septicemia secondary to bacterial infection. The diagnosis of cnf1⁺ E. coli was made based on reported clinical signs, including respiratory duress, feeding intolerance and vomiting, weight loss, lethargy, and seizures. Overall, the presence of cnf1⁺ E. coli led to rapid tissue destruction and health decline in the affected puppies. This exemplifies why infection by these bacteria requires immediate diagnosis and treatment.

During the 2013 outbreak at MSU, puppy mortality occurred within 1 week following birth. Exposure to the cnf1⁺ E. coli likely took place during parturition. In canines as well as humans, contamination with rectal and vaginal bacteria from the mother serves as the predominate infection source in neonates.^2,14–16 This is likely the case at MSU as well, based on the rectal and vaginal results from the dams who experienced litter death (Table 3), as well as the environmental samples taken from their respective whelping areas. With only the rectal sample being positive for cnf1⁺ E. coli, it is likely that pups born to female 5 were exposed to the bacteria after encountering the dam’s fecal matter in the whelping den. In a comparative study by Turchetto et al,¹⁷ exposure to cnf1⁺ E. coli from the dam’s feces resulted in the death of four out of five 12-day-old puppies. These neonates suffered from lethargy and weakness, as well as diarrhea from the time of whelping, signs that were also observed in the MSU puppies.

Adult carriers within the MSU vivarium remained asymptomatic, and no clinical signs were noted during the whelping of each affected 2013 litter, thereby raising no concern until puppies began displaying signs. Following the 2013 increased puppy mortality, uniform testing of all pregnant females was instituted; however, each PI chose to address these results differently. The inconsistency of PI treatment plans made assessment of the effectiveness difficult to measure. While widely accepted now, antimicrobial stewardship practices¹⁸ were not routinely practiced at the time of this outbreak. Current antimicrobial stewardship guidance for laboratory animal facilities state that investigators should reduce use, refine dose and route of administration, replace antimicrobials with nonantibiotic drugs where possible, review the need for antibiotics, and finally assume responsibility for the judicious use of antimicrobials.¹⁹ In 2023, the MSU canine research facility updated testing and treatment protocols for cnf1⁺ E. coli to include use of Visbiome Vet probiotic for pregnant females testing positive at 4-week gestation, followed by a retest at 7 weeks. If the subsequent test is positive, then appropriate antibiotics are administered.

Over time, an increase in resistance of cnf1⁺ E. coli to the commonly used antimicrobials was noted within the MSU canine research colony. These data suggest a particular increase in resistance of cnf1⁺ E. coli to cephalosporins, such as cefazolin, cefovecin, and cefpodoxime (Figure 3), an observation that was further confirmed, as the main antibiotic used to treat the original 2013 infections, cephalexin, is no longer effective against the most recent E. coli isolates. Further molecular testing would be required to fully understand the scope of antimicrobial resistance transfer within the MSU vivarium. Given that the dogs tested each year change depending upon which breeders are pregnant, this trend suggests that resistant strains are being transferred between individuals as susceptibility decreases.

Although the exact mechanism of recurrent infection of females in the research colony is unknown, several possible scenarios could explain the bacteria’s ability to spread. Although no E. coli was isolated from the whelping room or whelping boxes, the runs where females primarily live following whelping and weaning were not examined. These runs are cleaned and disinfected regularly, but it cannot be ruled out that some bacteria survive and serve as a source of future infection. Proper cleaning and disinfection are imperative in any healthcare environment. Surfaces should initially be cleaned and scrubbed to remove any excess amounts of dirt and gross material.²⁰ The mechanical action of scrubbing combined with the use of a detergent helps to reduce the number of potential pathogens.²¹ Once complete, cleaning should then be followed by a disinfection step. The disinfectant used, Fullsan II, is a quaternary germicidal cleaner that, at the proper dilution, can effectively eliminate E. coli after 10 minutes of contact time. Due to the passage of time, one cannot confidently know whether these procedures were regularly followed during the time of the outbreak.

In most animals, the initial line of defense against pathogenic microbes is the body’s passive immune response. At birth, puppies begin nearly absent IgG, approximating 5% the amount in a normal adult dog.² Research indicates that prior to suckling, neonatal pups possess little to no immune response protection,^14,22 and most of their protection is gleaned from the transfer of immunoglobulin from uptake of the mother’s milk. The level of passive immune transfer is negligible for newborn puppies but significantly increases 2 days following the start of colostrum intake.¹⁴ Given this delay in immunity buildup, bacterial invasion at this time allows ample opportunity for infection to spread with little resistance. Puppies at the MSU vivarium receive a formula supplement if trends of decreasing birthweight are observed, decreasing the volume of milk, as well as IgG antibodies, received from the dam. A previous study has shown that a bodily growth rate below −2.7% during the first 2 days of life indicates an IgG deficit in 87% to 96% of cases.²² Although specific immune level measurements were not taken, all puppies in this study met this negative growth rate standard (Table 1), which could suggest lowered transfer immunity.

Importantly, note the limitations faced when summarizing these cases. Puppies were submitted for necropsy as they passed, and those that passed over a short time frame were submitted as a cluster. Much of the tissue samples were pooled and tested in groups rather than as unique subjects. The pooling of data did have an impact on the amount of information available from necropsy reports. In addition, due to the passage of time and dealing with paper records, complete records for individuals were not necessarily available. The most individualized information came from notes regarding weight and recorded symptoms by caregivers. It is possible that information/paperwork was misplaced or became incomplete. Characterization of these isolates was limited to identification of only one virulence factor (cnf1). Assessment of additional factors would have provided a more complete understanding of the pathogenic nature of the E. coli isolated from the puppies. In addition, while there was an attempt to standardize treatment plans for pregnant/whelping females, each researcher housing dogs in the vivarium was able to have a unique treatment plan. This inconsistency impacted the ability to assess the overall prevalence of dogs testing positive for cnf1⁺ E. coli over time, but the impact is not able to be assessed. While the dogs in the vivarium are an isolated group, each PI has autonomy over their colonies, which can have different management plans.

Conclusions

Complete eradication of cnf1⁺ E. coli from the colony remains a difficult task due to the endemic nature of its presence within canine housing and breeding facilities. In facilities experiencing persistent cnf1⁺ E. coli infection, screening to identify carriers and posttreatment testing could help avoid bacteria passing between dogs. Rectal and vaginal culture swabs collected from every female are needed to understand how prevalent cnf1⁺ E. coli is within the colony, and new intakes should be tested during quarantine. Finally, antimicrobial stewardship protocols should be implemented and uniformity across PIs should be encouraged.