| Abstract | Za ovo istraživanje prikupljeno je 498 uzoraka fecesa kokoši s različitih peradarskih
farmi Bosne i Hercegovine. Nacijepljivanjem na podloge s dodatkom 1 mg/l cefotaksima
izdvojeno je ukupno 280 izolata bakterijske vrste Escherichia coli rezistentnih na cefotaksim.
Postupkom kombiniranih diskova u 74 izolata je fenotipski utvrđena tvorba betalaktamaza proširenog spektra i oni su uključeni u daljnje istraživanje. Disk difuzijskim
postupkom ispitana je njihova osjetljivost na slijedećih 12 antimikrobnih lijekova:
amoksicilin/klavulanska kiselina, cefoksitin, cefotaksim, ceftazidim, cefpodoksim, cefepim,
meropenem, enrofloksacin, ciprofloksacin, gentamicin, sulfametaksazol/trimetoprim i
tetraciklin. Osim na cefotaksim, svi izolati bili su rezistentni na cefpodoksim, a rezistencija na
ostale cefalosporine je bila statistički značajno niža. Na ceftazidim je bilo 54,1 %, na cefepim
50,0 % i na cefoksitin 48,6 % rezistentnih izolata. Najveći broj izolata bio je rezistentan na
amoksicilin s klavulanskom kiselinom (78,4 %), potom na tetraciklin (51,4 %). U osjetljivosti
izolata na fluorokinolone nije bilo statistički značajne razlike, njih 32,4 % je bilo rezistentno na
enrofloksacin, a 28,4 % na ciprofloksacin. Na sulfametaksazol/trimetoprim je bilo 18,9 %, a na
gentamicin 6,8 % rezistentnih izolata. Na meropenem nije bilo rezistencije. Šezdeset tri izolata
(85,1%) bila su rezistentna ili umjereno osjetljiva na tri ili više antimikrobnih lijekova iz
različitih kategorija prema čemu su procijenjeni kao multirezistentni izolati.
Kod 74 izolata lančanom reakcijom polimerazom ispitana je prisutnost gena blaCTX-M,
blaTEM i blaSHV. U 54 izolata (73,0 %) dokazana je prisutnost jednog ili više gena. Gen blaCTXM je dokazan u 33 izolata (61,1 %), gen blaTEM u 33 izolata (61,1 %) i gen blaSHV u jednom
izolatu (1,9 %). Kod 13 izolata (24,1 %) dokazana je istovremena prisutnost gena blaCTX-M i
blaTEM. Niti u jednom izolatu nije dokazana istovremena prisutnost sva tri gena (blaCTX-M,
blaTEM i blaSHV). Molekularna tipizacija gena blaCTX-M pokazala je da 32 od 33 izolata (97,0 %)
posjeduje gen blaCTX-M-1, a jedan od 33 izolata (3,0 %) posjeduje gen blaCTX-M-15. |
| Abstract (english) | Introduction: Escherichia coli (E. coli) is a Gram-negative, rod-shaped bacterium
found in the gastrointestinal tract of warm-blooded animals and humans as part of the normal
gut microbiota, but is also one of the most common causes of bacterial infections in humans
and animals. E. coli strains can be classified into two groups: commensal strains and pathogenic
strains that cause intestinal and extraintestinal infections in healthy individuals. In contrast to
commensal strains, intestinal pathogenic E. coli strains are rarely found in the normal intestinal
microbiota of healthy hosts. They appear to be essentially obligate pathogens that cause
gastroenteritis or colitis when ingested in sufficient amounts. Six pathotypes of intestinal
pathogenic E. coli strains are recognized: enteropathogenic (EPEC), enterohemorrhagic
(EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC), and
diffusely adherent (DAEC). Extraintestinal strains of E. coli (ExPEC) are usually found in the
normal gut microbiota as commensals but can cause various infections. These include
uropathogenic E. coli (UPEC) isolated from humans and animals with urinary tract infections,
neonatal meningitis-associated E. coli (NMEC) isolated from human neonates, septicemia
associated E. coli (SePEC), and also avian pathogenic E. coli (APEC) responsible for
colibacillosis in poultry.
E. coli is excreted with faeces and can survive for weeks or months in faecal particles,
dust and water, and enter the environment via faeces and wastewater treatment plants.
Pathogenic strains of E. coli can be transmitted from animals to humans through the food chain
and pose a direct threat to both the poultry industry and human health as they can lead to
difficult-to-treat infections. For the treatment of infections caused by E. coli, the most
commonly used classes of antimicrobials include beta-lactam antibiotics, fluoroquinolones,
aminoglycosides, sulfonamides, tetracyclines, and nitrofurantoin. Beta-lactam antimicrobials
are among the clinically most important antimicrobials used to treat bacterial infections in both
human and veterinary medicine. Because of their broad spectrum and low toxicity, they are
currently the most widely used class of antimicrobial agents to treat infections caused by E. coli strains. However, the widespread and often inappropriate use of antimicrobials has led to the
emergence of resistant bacterial isolates.
The predominant mechanism of beta-lactam resistance in E. coli is the production of
beta-lactamases, which inactivate antibiotics by hydrolyzing the beta-lactam ring. To date,
many different beta-lactamases have been identified, with extended spectrum β-lactamases
(ESBLs) being the most important. ESBLs are enzymes that can hydrolyze penicillins,
cephalosporins, and monobactams, but not cephamycins or carbapenems, and are inhibited by
beta-lactamase inhibitors such as clavulanic acid. There are three major ESBL families: TEM,
SHV, and CTX-M. The predominant and clinically most important ESBLs belong to the CTXM family, followed by the TEM and SHV families, which are found worldwide. The original
ESBL enzymes were variants of TEM and SHV variants that had amino acid substitutions that
resulted in a change in their substrate profile to include the extended-spectrum cephalosporins.
CTX-M enzymes originated from the Kluyveraa ascorbata chromosomal AmpC enzyme. Most
variants can be classified into five groups based on sequence homologies: CTX-M-1, CTX-M-
2, CTX-M-8, CTX-M-9, and CTX-M-25.
ESBLs are often encoded by genes located on plasmids, allowing them to spread among
bacteria, and they also carry genes for resistance to other classes of antimicrobial agents,
causing bacterial strains to become multidrug-resistant (MDR). Multidrug-resistant bacterial
strains are resistant to at least one agent from three or more antimicrobial drug classes. Such
strains of bacteria are of great public health concern, since multidrug-resistant bacterial
infections are difficult to treat as there are few or no treatment options.
Antimicrobial resistance has become a rapidly growing public health concern
worldwide and for this reason the World Health Organization (WHO) included antimicrobial
resistance as one of the top ten global health threats in 2019. The global distribution of ESBLpositive isolates is of great concern as these isolates are found in increasing numbers in
livestock, including poultry, retail meat and pets. The presence of ESBL-producing E. coli in
food animal production systems is of public health concern as it can be transmitted to humans
trough the food chain. Also, the presence of ESBL-positive isolates in companion animals can
pose a potential public health risk, as animals can serve as reservoirs of resistant bacteria for
humans.
Because of the limited therapeutic options and the risks associated with the acquisition
of new resistance mechanisms, ESBL-producing strains pose a major challenge for
microbiologists and clinical therapists. Therefore, laboratory detection of resistant strains prior
to the use of an antimicrobial agent in human and veterinary medicine is of great importance.
In addition, continuous education on the rational use of antibiotics is necessary to prevent the
spread of resistant and multidrug-resistant bacteria.
Today, the prevalence of food-producing animals carrying E. coli producing CTX-M
type ESBL has reached worryingly high levels. Food safety and the fight against antibiotic
resistance are particularly relevant in the One Health approach. Keeping this problem under
control requires constant monitoring of the evolution of the ESBL situation and the application
of an interdisciplinary One Health approach.
The aim of this study is to phenotypically confirm the presence of ESBL production in
E. coli isolates using combined disk diffusion method containing cefotaxime and ceftazidime
with and without clavulanic acid. A further aim is to detect the presence of the blaCTX-M gene
and to investigate the presence of the blaTEM and blaSHV genes in E. coli isolates. Also to identify
blaCTX-M genes for CTX-M- positive isolates.
Material and methods: In 2016 and 2017, a total of 498 faecal samples were collected
from different poultry farms in Bosnia and Herzegovina. Samples were cultured on MacConkey
agar supplemented with 1 mg/l cefotaxime after pre-enrichment with Tryptic Soy Broth
containing 1 mg/l cefotaxime. In this way, the selection of cefotaxime resistant isolates was
carried out.
The identification of E. coli was performed according to the procedure described by
Markey et al. (2013). All cefotaxime-resistant isolates were identified by standard
bacteriological culture and confirmed by several biochemical tests. E. coli colonies on
MacConkey agar are light pink, indicating acid production due to fermentation of lactose.
Colonies were identified biochemically as indole positive and oxidase negative using indole
and oxidase utilization assays.
After routine bacteriological analysis, all cefotaxime-resistant E. coli isolates were
screened for ESBL production using a combined disk diffusion method using cefotaxime and ceftazidime disc with and without clavulanate. Klebsiella pneumoniae ATCC 700603 was
included in the study as an ESBL positive control strain and E. coli ATCC 25922 as an ESBL
negative control strain. The diameters of the zones of inhibition were interpreted according to
the criteria recommended in the Clinical and Laboratory Standards Institute document (CLSI,
2018). An increased zone size in the presence of clavulanate of five millimeters or more was
considered indicative of ESBL production. All isolates that were phenotypically positive for
ESBL production were included in further studies.
Identification of the isolates was performed using the API 20 E identification system.
Antimicrobial susceptibility was determined using the standard disk diffusion method
for 12 antimicrobials: amoxicilin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime,
cefpodoxime, cefepime, meropenem, enrofloxacin, ciprofloxacin, gentamicin, trimethoprimsulfamethoxazole and tetracycline. Inhibition zone diameters were interpreted according to the
criteria recommended in the documents of the Clinical and Laboratory Standards Institute
(CLSI, 2015; CLSI, 2018).
All isolates phenotypically confirmed to be ESBL-producing E. coli were subjected to
molecular analysis using multiplex PCR screening to detect blaCTX-M, blaTEM and blaSHV genes.
Additional PCR was performed for blaCTX-M positive isolates with primers amplifying a 948 bp
PCR gene fragment for sequencing. PCR products were sequenced by Macrogen Europe,
Amsterdam, The Netherlands and the results were analyzed using the Basic Local Alignments
Tool (BLAST).
For statistical analysis STATISTICA v.14.0. (Statistica, Tibco, 2020.) program was
used. Fisher's exact test was used to compare statistically significant differences in the presence
of individual genes, including statistically significant differences in susceptibility among
isolates to antimicrobials from the same class.
Results: A total of 498 faecal samples were collected from several different poultry
farms in Bosnia and Herzegovina. A total of 280 cefotaxime-resistant E. coli isolates (56.2%)
were isolated. The prevalence of phenotypically confirmed ESBL-producing E. coli isolates in
poultry faeces was 14.9%. Based on the disk diffusion assay, all tested isolates showed
resistance to cefpodoxime (100.0%) in addition to resistance to cefotaxime. The highest resistance was observed to amoxicillin-clavulanic acid (78.4%), followed by ceftazidime
(54.1%). Resistance to tetracycline was 51.4%, followed by cefepime (50.0%) and cefoxitin
(48.6%). Resistance to enrofloxacin was 32.4% and to ciprofloxacin 28.4%. The lowest
resistance was observed to trimethoprim-sulfamethoxazole (18.9%) and there were only 6.8%
of isolates resistant to gentamicin. All isolates tested were susceptible to meropenem (100.0%).
Multidrug resistance was found in 85.1% of the isolates. Isolates were significantly more
resistant to cefotaxime and cefpodoxime (Fisher's exact test, p < 0,05). There was no
statistically significant difference between fluoroquinolones.
Among 74 phenotypically confirmed ESBL-producing E. coli isolates, 54 isolates
(73.0%) were positive for tested ESBL genes. Among them, 33 isolates (61.1%) carried the
blaCTX-M gene, 33 (61.1%) carried the blaTEM gene and only one isolate was positive for the
blaSHV gene (1.9%). The coexistence of blaCTX-M and blaTEM genes within the same isolate was
detected in 13 (24.1%) isolates, while none of the isolates tested showed the coexistence of
blaCTX-M, blaTEM and blaSHV genes. Sequence analysis of the blaCTX-M gene revealed that 32
(97.0%) of the isolates carried the blaCTX-M-1 gene and only one isolate (3.0%) carried the
blaCTX-M-15 gene. Of 74 isolates analyzed, 20 (27.0%) isolates had no ESBL genes.
Conclusions: The concentration of 1 mg/l cefotaxime as a selective antimicrobial agent
is low for the selection of ESBL-producing E. coli isolates.
Of 498 poultry faecal samples, 54 of the E. coli isolates (10.8%) were positive for ESBL
genes tested. These isolates can be the source of resistance genes and pose a public health threat.
In this study, the blaCTX-M-1 gene was the most prevalent, found in 32 out of 33 isolates
(97.0%). This result suggests that isolates from poultry are not involved in human infections in
Bosnia and Herzegovina, since among human isolates the most common gene variant is blaCTXM-15.
Of 74 isolates analyzed, 63 (85.1%) were found to be multidrug resistant, likely as a
result of the widespread and inappropriate use of antimicrobials in the poultry industry. |
