Efficiency of different enrichment media in the detection of gram negative bacilli in human, animal and environmental samples for

antimicrobial susceptibility tests

1,2

2,3

D. Duarte , J. D. Palmeira , H. M. N. Ferreira

1

First Degree in Biochemistry, Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal.
2
Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Portugal.
3
REQUIMTE, University of Porto, Portugal

Introduction

Methods
Methods

Enterobacteriaceae family consists of gram-negative facultative anaerobic bacteria that are widely
distributed and found in soil, water, vegetation and the intestinal tract of animals. [1] They are
causing agents of urinary tract and bloodstream infections, pneumonia and various intraabdominal infections which makes their detection, isolation and purification very important.[2]
Antibiotic resistance in Enterobacteriaceae is also a significant problem since over the past few
decades, the number of human infections caused by antimicrobial resistant bacteria increased
which can make treatment of these diseases more difficult.[3] There are several different enrichment media for the growth of gram negative bacilli which vary in their composition , but there are
no studies published that support which one should be used according the isolate. Current conventional methods for detection of Enterobacteriaceae involve selective enrichment followed by
plating onto selective media. Although enrichment media and selective agars have been continually developed during the last two decades [4], there are few publish studies that correlate which enrichment media should be used according the nature of the isolate.

All samples were measured (100mL of water by membrane filtration, 3g for sands, 1g for the other
ones), incubated at 37 C overnight with 40 mL of enrichment broth (EB). Cultures were diluted
from 1:10 to 1:100 000 and spread in a solid selective for Gram negatives and differential culture media (MacConkey), to detect different colony types. Colony-forming units were analysed in order to
differentiate lactose fermenting and non-fermenting bacteria morfologies, from the MacConkey
agar plates with better colony dispersion.

This study develops a methodology for the culture and isolation of gram negative
bacteria by comparison of performance of five enrichment broths: tryptic soy
broth (TSB), brain heart infusion broth (BHI), brilliant green, peptone water and buffered peptone water, for the detection of gram negative bacteria,
namely coliforms, in 35 samples of different origins: human, animal (domestic and
bovine) and environmental (water, sand and mud).

37
EB

24h

Samples were suspended in TSB and inoculated in MacConkey agar with ciprofloxacin, cefotaxime
and meropenem. Colonies were randomly selected and tested for antimicrobial susceptibility to 9
antibiotics by disk-diffusion method on Mueller-Hinton Agar according to the Clinical Laboratory
Standards Institute.The following disks were used: amoxicillin (AML) (10 g), amoxicillin/
clavulanic acid (AUG) (20/10 g), cefoxitin (FOX) (30 g), cefepime (FEP) (30 g), cefotaxime (CTX) (30 g), meropenem (MRP) (10 g), ciprofloxacin (CIP) (5 g), tetracycline (TE)
(30 g) and trimethoprim/sulfamethoxazole (SXT) (1.25/23.75 g). E. coli ATCC was used as
quality control. Isolates were classified as sensitive, intermediate or resistant according to the CLSI
recommendations after 24 h incubation at 37C.

TSB + sample

Results
Results

Graph 1. Lactose non-fermenting colony forming-units from the MacConkey agar plates with better
colony dispersion for different isolates.

Analysing sand isolates, BHI proved by far to be the most

appropriate enrichment medias for the enhancement of
lactose non-fermenting bacteria followed. For water, mud,
dog, cat and human samples, the most suitable enrichment media for the growth of those was peptone water
and buffered peptone water; however, for water and dog,
the buffered media favoured most the spread of bacteria
than the other one. Bovine isolates presented a prevalente
growth in BHI.

Graph 2. Lactose fermenting colony forming-units from the MacConkey agar plates with better
colony dispersion for different isolates.

By graphs analysis, it can be seen that BHI enhanced most

the growth of lactose fermenting bacteria in sand and cat isolates, although Brilliant Green can also be considered a suitable enrichment media for those bacteria in sand samples. For
water, mud and bovine samples, the most suitable enrichment media intended for this purpose was Brilliant Green.
However, for dog and human isolates, Peptone Water and TSB
presented better results, respectively.

Graph 3. Resistance of cat, dog and sand isolates the tested antibiotics.

After susceptibility tests, it can be seen that cat isolates exhibited more resistance to amoxicillin and amoxicillin with
clavulanic acid; dog isolates showed resistance to previously
antibiotics with the addition of ciprofloxacin, cefotaxime,
tetracycline and trimethoprim/sufamethoxazole. In relation
to environmental samples (sands), they were more resistant
to amoxicillin and amoxicillin with clavulanic acid. All samples were more susceptible to meropenem and cefepime.

Figure 2. Culture medium MacConkey agar with a water isolate in Brilliant

Green. Note the absence of lactose non-fermenting bacteria and the high
number of lactose fermenting CFU. Dilution 1:100 000

Figure 1. Culture medium MacConkey agar with a water isolate

in buffered peptone water. Note the marked presence of lactose
non-fermenting bacteria. Dilution 1:100 000

Figure 3. Antibiotic susceptibility test of a sand isolate showing and

ESBL in culture medium Mueller-Hinton agar

Conclusions
Conclusions
All enrichment broths demonstrated capacity of increasing the bacterial mass of the samples; however it was found that TSB is an efficient enrichment medium without favouring the
growth of lactose fermenting or non-fermenting gram negative bacilli; Brilliant green showed a prevalent enrichment of lactose fermenting gram negative bacilli; BHI, peptone water and
buffered peptone water provided a significant increasing of lactose non-fermenting gram negative bacilli. It was also found that environmental samples are those who most need to be enriched, unlike human and animal samples, whose enrichment may be dispensed due to its huge natural flora. Animal and environmental samples showed the presence of multi-resistant
lactose fermenting Enterobacteriaceae, which is a worrying question in terms of public health. These results showed the effectiveness of TSB for detection of isolates resistant to antibiotics towards other enrichment broths in this specific approach.

References

Acknowledgements

[1] Blood R. M et al (1995) Media for total Enterobacteriaceae, coliforms and Escherichia Coli. Int. J. Food Microbiology, 26, 97-115
[2] Leonard AF, Zhang L, Balfour AJ, Garside R, Gaze WH (2015) Human recreational exposure to antibiotic resistant bacteria in coastal bathing waters. Environ Int. 2015 Mar, doi:
10.1012/j.envint.2015.02.013.
[3] Fryet J. G et al (2013) Genetic mechanisms of antimicrobial resistance identified in Salmonella enterica, Escherichi coli and Enterocccus spp isolated from U.S. food animals. Frontiers in Microbiology
[4] Baylis C.L et al (2000) Comparison of three enrichment media for the isolation of Campylobacter spp. from foods. Journal of Applied Microbiology, 89, 884-891

Helena Neto, Josman Palmeira, Marisa Pinto