Elizabethkingia anophelis: The Upper Midwest Scourge

A multistate outbreak of infections caused by a bacterium called Elizabethkingia anopheles is being investigated by the Centers for Disease Control and Prevention (CDC), the Wisconsin Department of Health Services (WDHS), the Michigan Department of Health and Human Services (MDHHS) and the Illinois Department of Public Health (IDPH) (Table1).

* These are cases that tested positive for Elizabethkingia, but will never be confirmed as the same strain of Elizabethkingia anophelis because the outbreak specimens are no longer available to test.

Wisconsin

• November 1, 2015 – April 8, 2016, the Wisconsin Department of Health Services (WDHS) has continued to investigate an outbreak of bloodstream infections caused by a bacterium of the genus Elizabethkingia.
• There have been 57 confirmed cases of Elizabethkingia anophelis infections reported to the WDHS resulting in 19 fatalities. There have been 18 deaths among individuals with confirmed Elizabethkingia anophelis infections and an additional 1 death among possible cases.
• January 5, 2016, Wisconsin set-up statewide surveillance after first being notified by the CDC of six potential cases occurring between December 29, 2015 and January 4, 2016.
• The majority of individuals in Wisconsin acquiring this infection are over the age of 65 and all have a history of at least one underlying serious illness. At this time, the source of these infections is unknown. The WDHS is working diligently to contain this outbreak.
• Wisconsin counties affected include Columbia, Dane, Dodge, Fond du Lac, Jefferson, Milwaukee, Ozaukee, Racine, Sheboygan, Washington, Waukesha and Winnebago.

CDC

• On January 20, 2016, a nationwide call for cases was issued via the Emerging Infections Network.
• On March 2, 2016, a nationwide call for cases was issued via the Epidemic Information Exchange system, also known as Epi-X.
• These alerts asked states to look for any infections similar to the ones reported in Wisconsin and to send isolates from any potential cases to the CDC for testing to determine if they match Elizabethkingia anopheles, the bacteria causing infections in Wisconsin.

Michigan

• On February 8, 2016, MDHHS sent a state health alert asking providers and laboratories to review records for Elizabethkingia specimens identified since January 1, 2014 in response to the outbreak of Elizabethkingia infections in Wisconsin.
• On February 29, 2016, the MDHHS Bureau of Laboratories received an Elizabethkingia specimen from a recently submitted blood culture isolate from a Michigan resident. This blood culture isolate was forwarded to the CDC for additional testing.
• On March 11, 2016, the MDHHS was notified by the CDC of the Elizabethkingia anophelis match.  The West Michigan case resulted in the death of an older adult with underlying health conditions.

Illinois

• On February 10 and March 29, 2016, the IDPH sent alerts to hospitals requesting they report all cases of Elizabethkingia and save any specimens for possible testing at public health laboratories.
• To date, only one isolate from Illinois has matched Elizabethkingia anopheles resulting in one death.

The majority of the patients who have had Elizabethkingia infections as part of this outbreak are over the age of 65 years and all have had serious underlying health conditions. The majority of the infections identified to date have been bloodstream infections but some patients have had Elizabethkingia isolated from other sites such as their respiratory systems or joints.  It has not been determined whether the deaths associated with this outbreak were caused by the bacterial infection, the patients’ underlying health conditions or both. Most outbreaks associated with Elizabethkingia are healthcare-associated. There are few reports of community-acquired infections.

Elizabethkingia infections are often difficult to treat with antibiotics.  These bacteria are resistant to many of the antibiotics healthcare providers use to treat infections. Early recognition of the bacteria is critical to ensure patients receive appropriate treatment and increase the probability of good outcomes. The signs and symptoms of illness that can result from exposure to the bacteria include fever, shortness of breath, chills or cellulitis. Confirmation of the illness requires a laboratory test.

The CDC is continuing to work with the WDHS, MDHHS and IDPH to identify the source of the bacteria and develop ways to prevent these infections. The CDC is assisting with testing samples from a variety of potential sources, including healthcare products, water sources and the environment. To date, none of these have been found to be a source of the bacteria. 

• 1959 - American bacteriologist Elizabeth O. King was studying unclassified bacteria associated with pediatric meningitis at the CDC. She isolated an organism (CDC group IIa) that she named Flavobacterium meningosepticum (Flavobacterium means "the yellow bacillus" in Latin; meningosepticum means "associated with meningitis and sepsis").
• 1994 - Flavobacterium meningosepticum was reclassified in the genus Chryseobacterium and renamed Chryseobacterium meningosepticum (chryseos means "golden" in Greek; Chryseobacterium means a golden/yellow rod similar to Flavobacterium).
• 2005 - A 16S rRNA phylogenetic tree of Chryseobacteria showed that C. meningosepticum along with C. miricola (which was reported to have been isolated from the Russian space station Mir in 2001 and placed in the genus Chryseobacterium in 2003) were similar to each other but outside the tree of the rest of the Chryseobacteria. They were then placed in a new genus Elizabethkingia named after the original discoverer of F. meningosepticum.
• 2008 - Green et al described the first case of Elizabethkingia miricola sepsis in an immunocompromised host.
• 2011 - Elizabethkingia anophelis is a bacterium isolated from the midgut of Anopheles gambiae mosquitoes originating from MacCarthy Island, The Gambia.
• 2013 - E. anopheles was reported as a human pathogen in the Central African Republic where a clinical case of neonatal meningitis was reported. A nosocomial outbreak was also reported in an intensive care unit (ICU) in Singapore. In both clinical cases, multidrug resistance was reported and the isolates were resistant to a wide array of antibiotics.
• 2014 – A study showed that some Elizabethkingia infections that had been attributed to Elizabethkingia meningoseptica were instead caused by Elizabethkingia anophelis.
• 2015 - An outbreak centered in Wisconsin began in early November 2015, with 48 individuals confirmed infected in 12 counties with at least 18 deaths by March 9, 2016. Four new cases were documented just in the week of March 2 – 9, 2016. 

The genus Elizabethkingia currently includes four species (Table 2):

• E. meningosepticum (or E. meningoseptica)
  • Binomial Name: Elizabethkingia meningseptica
  • Synonyms: Chryseobacterium meningosepticum, Flavobacterium meningosepticum, Elizabethkingia meningosepticum and CDC Group IIa
  • E. meningosepticum causes neonatal sepsis and infections in immunocompromised individuals.
• E. miricola
  • Binomial Name: Elizabethkingia miricola
• E. endophytica
  • Binomial Name: Elizabethkingia endophytica
• E. anopheles
  • Binomial Name: Elizabethkingia anopheles

E. anophelis has a circular genome of 4,369,828 base pairs and 4,141 predicted coding sequences. 16S rRNA gene sequence analysis revealed that E. anopheles showed 98.6% sequence similarity to that of E. meningoseptica and 98.2% similarity to that of E. miricola.

DNA-DNA hybridization experiments with E. meningoseptica and E. miricola gave relatedness values of 34.5 % (reciprocal 41.5 %) and 35.0 % (reciprocal 25.7 %), respectively. DNA-DNA hybridization results and some differentiating biochemical properties indicate that species R26(T) represents a novel species, for which the name E. anophelis was proposed.

The draft genomes were annotated using the National Center for Biotechology Information (NCBI) Prokaryotic Genome Automatic Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/), which predicted 3,687 protein-coding sequences (CDS) and 44 RNA genes in R26(T) and 3,648 CDS and 38 RNA genes in Ag1. Strikingly, 112 protein features were identified in the category “resistance to antibiotics and toxic compounds”. This included drug efflux/transport (36 features); resistance to β-lactam antibiotics, fluoroquinolones and heavy metals (28, 4 and 25 features, respectively) and 19 additional features involved in resistance to a diverse set of antibiotics. The large genetic capacity against various antibiotics is consistent with the observation that E. anophelis has natural antibiotic resistance to several antibiotics.

E. anophelis is a bacterial species in the family Flavobacteriaceae. The bacterium is a slightly yellow-pigmented, non-motile, non-spore-forming, gram-negative, rod-shaped cell. E. anophelis has two growth optima at 30 - 31 °C and 37 °C. It is a dominant resident in the mosquito gut of the malaria vector mosquito, Anopheles gambiae, and also a human pathogen. The possibility of a role for mosquitoes in the maintenance and transmission of E. anophelis remains unclear.

E. anophelis utilizes complex carbohydrates (glycans) in its metabolism with starch-utilization systems (Sus) including proteins:

SusD (glycan-binding protein)

SusC (Ton-B dependent transporter)

SusE/SusF (carbohydrate-binding proteins without enzyme activity)

SusA, SusB, SusG (enzymes for polysaccharide deconstruction)

SusR (an inner membrane-associated sensor-regulator system for transcriptional activation of Sus genes).

The major fatty acids of E. anophelis, strain R26(T) were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 4 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c/t). Strain R26(T) contained only menaquinone MK-6 and showed a complex polar lipid profile consisting of diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid and unknown polar lipids and glycolipids.

The bacterium produces several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. Numerous TonB-dependent transporters (TBDTs) with various substrate specificities help the bacterium to utilize polymers. E. anophelis has well-developed systems for scavenging iron and stress response. The bacterial TBDTs are specialized elaborate machinery for active uptake of rare but essential nutrients and other substrates, such as iron complexes, vitamin B12, nickel, carbohydrates and colicin. To energize the transport process, TBDTs interact with the TonB complex, a cytoplasmic transmembrane assembly of the proteins ExbB and ExbD, which couples with the TonB in periplasm.

The cell also produces efflux pumps and β-lactamases that give the bacterium broad antibiotic resistance. RNA-sequencing-based transcriptome profiling indicates that expressions of genes involved in synthesis of a yersinibactin-like iron siderophore and heme utilization are highly induced as a protective mechanism toward oxidative stress caused by hydrogen peroxide stress. E. anophelis produces OxyR regulon and antioxidants that may provide defense against the oxidative stress that is associated with blood digestion in mosquitoes. One study showed that hemoglobin facilitates the growth, hydrogen peroxide tolerance, cell attachment and biofilm formation of E. anophelis and that siderophore production and heme uptake pathways might play essential roles in stress response and virulence.

E. anophelis is a dominant bacterial species in the gut ecosystem of the malaria vector mosquito, Anopheles gambiae. Like some Bacteroidetes, E. anophelis possesses polysaccharide utilization loci (PUL), which suggests the genetic capability to utilize various plant polysaccharides. This implies an intriguing ecological connection with the nectar and plant sap feeding behavior of mosquitoes in nature. The predominance of E. anophelis in the sugar fed gut of mosquitos and the possession of numerous Sus-like loci and GHs suggest that the bacterium may be capable of utilizing plant cellulose in the diet and could potentially be of benefit for the mosquito carbohydrate metabolism. The interactions among antibiotic-producing and resistant bacteria may be one of the determinants that shape and stabilize the community structure in the mosquito gut. The bacterium also displays hemolytic activity and encodes several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. At the same time, the OxyR regulon and antioxidant genes could provide defense against the oxidative stress that is associated with blood digestion. The genome annotation and comparative genomic analysis revealed functional characteristics associated with the symbiotic relationship with the mosquito host.

Little information exists about the pathogenesis of Elizabethkingia infections in general. Colonization of the respiratory tract with Elizabethkingia infections usually precedes invasive infection particularly in high risk populations such as neonates and adults in ICUs. Early and late onset neonatal meningitis is hypothesized to have been preceded by colonization of an infant at the time of delivery since this has been described for other common neonatal pathogens. However, this hypothesis has not been studied.

Elizabethkingia is a genus of bacteria commonly found in the environment and has been detected in soil, river water and reservoirs. They are not normally present in human microflora. It rarely causes infections. Elizabethkingia, though, are opportunistic pathogens preferentially affecting individuals with compromised immune systems or serious underlying health issues. These pathogens are associated with a high mortality rate.

• 2013 - E. anophelis was reported as a human pathogen in Central Africa in a clinical case of neonatal meningitis and an outbreak was also seen in an ICU in Singapore. In both clinical cases, multidrug resistance was reported and the isolates were resistant to a wide array of antibiotics.
• 2014 - Teo et al. highlighted that E. anophelis is an emerging bacterial pathogen for hospital environments. It has been associated with neonatal meningitis and nosocomial outbreaks. Its transmission route remains unknown.
• 2015 - Lau et al. used rapid genome sequencing and comparative genomics to investigate three cases in Hong Kong of E. anophelis sepsis involving two neonates who had meningitis and one neonate’s mother who had chorioamnionitis. Evidence was provided for perinatal vertical transmission from a mother to her neonate. The two isolates from these patients, HKU37 and HKU38, shared essentially identical genome sequences. In contrast, the strain from another neonate (HKU36) was genetically divergent, showing only 78.6% genome sequence identity to HKU37 and HKU38, thus excluding a clonal outbreak. Comparison to genomes from mosquito strains revealed potential metabolic adaptations in E. anophelis under different environments. Maternal infection, not mosquitoes, was the most likely source of neonatal E. anophelis infections. The pathogenic and multiresistant nature of the bacteria prompted investigations of the vector potential of mosquitoes for E. anophelis transmission to humans. The antibiotic resistance might have consequences for future work with E. anophelis. These case reports raised a concern regarding whether or not mosquitoes can pass E. anophelis and E. meningoseptica to humans in clinical situations and when handling mosquitoes in research. Further investigation is required to evaluate these potential risks.

Researchers do not know exactly how E. anophelis is transmitted. They suspect individuals get sick by coming into contact with the bacteria in hospitals or through contaminated water. They are also finding that the bacteria can be passed from mothers to babies.

Researchers in Wisconsin are reasonably certain that groundwater is not the source. The CDC is also assisting Wisconsin with testing of samples, including health care products, water sources and the environment. "To date, none of these have been found to be a source of the bacteria."

Risk factors which should raise suspicion for Elizabethkingia infections including E. meningisepticum and E. anophelis include:

• Age
  • Very young
  • Over the age of 65 (median age 77)
• Patients with underlying serious health conditions that compromise their immune systems such as:
  • Malignancy
  • Diabetes mellitus
  • Chronic renal disease or end-stage renal disease on dialysis
  • Alcohol dependence
  • Alcoholic cirrhosis
  • Immunosuppressive treatment

General symptoms of E. anophelis are very non-specific and may mimic many other infections. Generalized symptoms may include:

• Fever/chills
• Shortness of breath
• Rashes 
• Cellulitis, a redness and swelling from a skin infection that may feel hot and tender to the touch (most commonly on the lower extremities)
• Sepsis/Septic shock

The optimal antimicrobial therapy for any Elizabethkingia infection is difficult to determine. Empiric therapy is based on the unusual susceptibility pattern, the availability of effective antimicrobials, the age of the patient, clinical presentation and reported experience.

Definitive therapy should rest upon the susceptibility results of the isolated organism. Important considerations to remember when managing these infections include:

• Disk diffusion techniques are not reliable and treatment failures can occur if treatment is based on disk results.
• Antimicrobial susceptibilities should be confirmed using a quantitative method.
• Acquired antimicrobial resistance can develop during therapy so repeat cultures and susceptibility testing is recommended if the patient remains culture positive, does not improve or deteriorates.

Elizabethkingia infections are bacterial and treated with antibiotics. The problem is the bacteria often does not respond to the standard antibiotics. The antibiotics that work against Elizabethkingia infections are usually surprising and the last ones physicians would prescribe. Initial identification of the bacteria (as gram negative) would lead medical professionals to likely empirically treat with drugs that might not work.

Elizabethkingia infections are usually resistant to many antibiotics that commonly treat gram negative bacteria such as:

• Aminoglycosides
• β-lactam drugs, including carbapenems
• Extended spectrum β-lactamase (ESBL) agents
• Metallo-β-lactamase agents

These are the antibiotics that are often thought to be the biggest "guns" against bacteria. However, Elizabethkingia infections are gram negative but seem to respond to some drugs used for gram-positive bacteria – like Vancomycin which is usually solely able to treat gram positive infections.

There is less experience with treating E. anophelis, but prior to this outbreak the bacteria was shown, without prior known antibiotic exposure, to be resistant to:

• Ampicillin
• Chloramphenicol
• Kanamycin
• Streptomycin
• Tetracycline

In the Wisconsin outbreak, E. anophelis appears susceptible to:

• Bactrim (trimethoprim/sulfamethoxazole)
• Flouroquinolones like levofloxacin or ciprofloxacin
• Zosyn (piperacillin/tazobactam)
• Combination therapy is usually recommended – as is the potential addition of Vancomyin.

No vaccines are currently available to prevent any Elizabethkingia species.

Clinical studies have demonstrated the importance of rapid instigation of epidemiological investigation, while also ensuring that fundamental infection control procedures are in place during an outbreak and to prevent an outbreak.

Examples of infection control procedures that may be implemented include:

• Patients known to be infected or colonized via positive cultures with Elizabethkingia should be placed in isolation following the institutional guidelines for multiple antibiotic-resistant organisms.
  • If a patient with a known or suspected Elizabethkingia infection is to be transferred, the receiving facility should be notified of the patient’s history of known or suspected Elizabethkingia infection upon transfer.
  • In long-term care and assisted living facilities, residents can be managed according to the DPH Guidelines for the Prevention and Control of Antibiotic-Resistant Organisms in Healthcare Settings.
• All healthcare facilities and staff should use contact precautions in addition to standard precautions when managing patients with Elizabethkingia infections.
  • CDC hand hygiene guidelines in health-care settings
  • CDC environmental infection control guidelines in health-care facilities
• In-service training should be implemented to reemphasize handwashing and contact precautions to all staff.
  • Use of alcohol based hand rub after washing of hands should be emphasized.
• Measures that have been used to eradicate Elizabethkingia outbreaks in healthcare settings have included:
  • Changing the prescribing policy for empiric antibiotics
  • Restriction of further admissions
  • Restriction of staff exchange between wards
  • Scrubbing units and wards thoroughly using two disinfectants (hypochlorite solution and isopropanol spray) with special emphasis on objects containing or in contact with water
  • Particularly in ICUs, frequent disinfection of electrical buttons, computer keyboards, phones, doorknobs and Ambu bags
• The infection control team, knowing gram-negative bacteria may have an inanimate reservoir, may investigate the:
  • Hospital sinks
  • Repairing, cleaning, super chlorination, isolation of the water tanks from all the hospital feeder tanks and changing the sink taps
  • Handling of respiratory equipment, especially nebulizers with a reservoir
  • Discarding of opened creams, ointments, sterile water and hand-washing solutions
  • Strict supervision of the preparation process of intravenous solutions
• To detect the source of an outbreak of Elizabethkingia infections, it is important to obtain cultures from food and infant formulas, wet areas, dry surfaces, equipment and the hands of healthcare workers who have contact with patients. Obtaining cultures on a periodic basis is necessary.

In conclusion, surveillance for the reservoir and maintenance of rigorous infection control measures are essential to control Elizabethkingia outbreaks in healthcare settings.

In all the cases in the United States, whether the bacteria caused or contributed to the deaths remains unclear. Any Elizabethkingia species can be deadly. The mortality rate from Elizabethkingia is usually around 20% although some outbreaks have resulted in the deaths of more than half of those infected.

Patients with Elizabethkingia infections who have high risk factors have an increased mortality. Several predictors of poor outcome include:

• Hypoalbuminemia
• Increased pulse rate at the onset of infection
• Central venous line infection 

Healthcare facility response measures should include:

• Immediately reporting the identification of any isolation of Elizabethkingia from any sterile site specimen (blood, cerebral spinal fluid, synovial fluid, pleural fluid or other sterile site) to the respective State Health Department.
• Faxing requested medical records (including face sheet) to the respective State Health Department.
• Some clinical laboratories use bacterial detection systems with software that is not updated to report Flavobacterium meningosepticum or Chryseobacerium meningosepticum as Elizabethkingia meningosepticum. Therefore, it is advised to report the detection of any isolate from a sterile site specimen that is identified as F. meningosepticum, C. meningosepticum or E. meningosepticum.
• Submit all Elizabethkingia isolates (or F. meningosepticum or C. meningosepticum) expeditiously to the respective State laboratory for confirmatory testing via the facility clinical microbiology laboratory.
• Cases should be reported to Public Health officials – as should cases caused by similar bacteria. In particular, other Elizabethkingia species or related bacteria should be reported as they may have been misidentified.

The past few years have seen an upsurge in the number of pathogens from primarily bacteria and viruses i.e., Elizabethkingia infections, Zika virus etc. which previously affected other countries populations. We have tended to focus our attention on “our own” diseases and less on other countries. Now, outbreaks arise and healthcare systems rush to stem the tide of the damage.  E. anophelis should remind us that pathogens abide by no international boundaries…all pathogens are a risk to all peoples worldwide.