Figure 1.3. Indirect Resistance.

Figure 1.4. PCR amplification of blaTEM-1, blaOXA-48and16S RNA genes. K. pneumoniae is abbreviated as KP,E. coli as EC and negative control as NEG. We tested if E.coli received the resistance genes OXA-48 and TEM-1 fromK. pneumoniae and for the presence of DNA using 16S RNA.K. pneumoniae showed both genes. E. coli did not receivethe OXA-48 gene but showed a band for TEM-1.The TEM-1negative control was DNA-free but showed a miniscule band.

Result I – E. coli protection

• To our best knowledge, this is the first study of IR in K.pneumoniae.

• Our results show K. pneumoniae can protect E. coli inco-culture.

• This may be via indirect resistance because blockingSecA stopped E. coli protection and SecA is involved inthe periplasmic transport of bacterial proteins.

• The faint band in the TEM-1 control suggests there iscross-contamination in the PCR, and we will need tovalidate this.

• E. coli may have received the TEM-1 gene• These results reveal an important role of K. pneumoniae

on antibiotic-resistance in polymicrobial communities.

I would like to thank Dr Helen McRobie and theMicrobiology department staff for their support andguidance throughout the course of my project.Contactkudzai.hwengwere@student.Anglia.ac.uk

ReferencesNicloff, H. and Andersson, D., 2016. Indirect resistance toseveral classes of antibiotics in cocultures with resistantbacteria expressing antibiotic-modifying or-degradingenzymes: Journal of antimicrobial chemotherapy, 71, pp.100-110.Chaudhary, A.S. Chen, W. Jin, J. Tai, P.C. and Wang, B.,2015. SecA: a potential antimicrobial target: FutureMedical Chemistry, (7), 8, pp.989.

Result III – SecA Inhibition

• We grew co-cultures of ampicillin-susceptibleEscherichia coli CSHO26 and ampicillin-resistantKlebsiella pneumoniae K17 in 100 μg/ml ampicillin totest for indirect resistance.

• We found that E. coli was protected from antibiotic-killing in co-cultures with K. pneumoniae.

• We showed that E. coli protection was blocked by RoseBengal, a known inhibitor for the general secretory(Sec) pathway.

K. pneumoniae is a Gram-negative rod-shaped bacteriumthat belongs to the Enterobacteriaceae family. Thepathogenic form can induce local and systemic infectionsin humans. Alarmingly, some strains of K. pneumoniae areresistant to nearly all lines of antibiotics, making it a globalhealth threat. Indirect resistance (IR) is the ability of anantibiotic-resistant bacteria strain to protect susceptiblebacteria without transferring genetic determinants. IR hasnot yet been described in K. pneumoniae. In this study weinvestigated if ampicillin-resistant K. pneumoniae couldprotect ampicillin-susceptible E. coli in co-culture. E. colisurvived at a lethal concentration of ampicillin in thepresence of K. pneumoniae. We then explored whetherthis was due to direct resistance (gene transfer) or indirect(antibiotic resistant bacterial protein secreted into theperiplasm or environment). We blocked the generalsecretory (Sec) pathway using Rose Bengal and this hassubsequently inhibited the protection of E. coli. Our resultssuggest both indirect and direct resistance might beinvolved in the protection of E. coli by K. pneumoniae.These findings require further validation but highlight theimportance of K. pneumoniae resistance in mixedinfections and present opportunities to study the effect ofK. pneumoniae on the evolution of resistance in co-habitating susceptible species.

Figure 1.1. Indirect resistance. β-lactamase is abacterial protein that degrades β-lactam antibiotics.Indirect resistance can be achieved by antibiotic resistantbacteria secreting β-lactamase into the environment.Adapted from Nicloff and Andersson (2016).

Summary

We investigated indirect resistance (IR) between K.pneumoniae and E. coli by growing bacteria on LB agarplates with or without ampicillin (100 µg/ml). Prior to thisexperiment, we measured the minimal inhibitoryconcentration and showed that K. pneumoniae is resistantto ampicillin < 1024 µg/ml, while E. coli was sensitive toampicillin > 32 µg/ml. We tested bacterial growth in sixconditions (Table 1). To determine indirect antibioticresistance, we dropped K. pneumoniae onto a spreadplate of E. coli in LB-agar supplemented with 100 µg/mlampicillin. All plates were incubated for 24-48 hours at 37˚C and pictures of colonies were taken (Figure 1.3.)Subsequently, K. pneumoniae and E. coli isolates werescreened for the antibiotic-resistance genes, blaOXA-48and blaTEM-1.Table 1. Test conditions to determine IR.

Method I – Indirect Resistance

Rose Bengal, a SecA inhibitor, was tested for its effectson E. coli protection by K. pneumoniae. SecA is anATPase motor that binds to SecYEG (transmembraneprotein complex). It attaches to the proteins to be exportedby the cell and through its ATPase activity facilitates thetranslocation of proteins into the periplasm (Figure 1.2). Ithas been shown to transport β-lactamases. E. coli and K.pneumoniae were co-cultured on a LB-agar platesupplemented with 100 µg/ml ampicillin culture and 50µg/ml of Rose Bengal. The plate was incubated for 24hours at 37 ˚C.

Figure 1.2. Sec-dependent translocation. Adapted from(Chaudhary et al, 2015).

Other logos here

Condition K. pneumoniae E. coli Ampicillin (100 µg/ml)

1 present absent absent2 present absent absent3 absent present absent4 absent present present5 absent absent absent6 present present present

K. pneumoniae 100 µg/ml ampicillin

K. pneumoniae and E. coli 100 µg/ml ampicillin

K. pneumoniaeNo ampicillin

E. coli 100 µg/ml ampicillin

No bacteriaNo ampicillin

Negative Controls

Positive Controls

Main Experimental Result

E. coliNo ampicillin

E. coli satellite colonies K. pneumoniae

1. 2. 3.

4. 5.

6.

SecA inhibition

K. pneumoniae and E. coli100 µg/ml ampicillin

50 µg/ml Rose Bengal

Figure 1.5. Rose Bengal inhibited E. coli protection. Inthe presence of Rose Bengal, K. pneumoniae survived butthere were no E. coli satellite colonies observable.

KPOXA-48

KPTEM-1

EC 16S RNA

DNA MWT ladder

kbp

0.51.01.5

NEGTEM-1

NEGOXA-48

NEG16S RNA

ECOXA-48

3.0

ECTEM-1

KP 16S RNA

Investigation into Indirect Antibiotic Resistance in Co-cultures of Klebsiella pneumoniae and Escherichia coli

Kudzai Hwengwere, Dr Helen McRobie, Department of Microbiology, Anglia Ruskin University, UK

Periplasm

Bacteria producing β-lactamase

Indirect resistance

β-lactamaseKey

β-lactam SecYEG

Method II – SecA inhibition

Abstract

Background – Indirect Resistance

Result II – Test for Gene Transfer

Conclusion

Acknowledgements