Occurrence and characteristics of extended-spectrum-ß-lactamase producing Escherichia coli (blaTEM-128) isolated from Mus musculus captured from a veterinary clinic and houses in Tunis, Tunisia.

Antimicrobial resistance in Enterobacteriaceae is a public health problem. Rodents, can be a potential vector for transmission of multidrug resistant bacteria between animals, humans, and environment. The aim of our study was to assess the level of Enterobacteriaceae present in the intestines of rats collected from different locations in Tunisia, then to determine their antimicrobial susceptibility profiles, to screen extended spectrum ß-lactamases-producing strains and determine the molecular mechanism of ß-lactams resistance. Between July 2017 and June 2018, 55 strains of Enterobacteriaceae were isolated from 71 rats captured in various locations in Tunisia. Antibiotic susceptibility testing was performed using the disc diffusion method. Genes encoding ESBL and mcr genes were investigated by RT-PCR, standard PCR and sequencing when these genes were found. Fifty-five strains of Enterobacteriaceae were identified. The overall prevalence of ESBL production found in our study was 12.7 % (7/55) of which two E. coli strains were DDST positive, one isolated from a house-caught rat and one from the veterinary clinic and harbored the blaTEM-128 gene. In addition, the other five strains were DDST negative and harbored the blaTEM gene, including three strains isolated from collective restaurant (n = 2: blaTEM-163; n = 1: blaTEM-1), one strain isolated from the veterinary clinic (blaTEM-82), and one strain isolated from a house (blaTEM-128). The results of our study suggest that rodents may play a role in the spread of antimicrobial resistant E. coli, highlighting the need to protect the environment and monitor antimicrobial resistant bacteria in rodents to prevent their spread to other wildlife and humans.

First Report of OXA-48 and IMP Genes Among Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Diarrheic Calves in Tunisia.

Antimicrobial resistance is one of the most serious threats to human and animal health. Evidence suggests that the overuse of antimicrobial agents in animal production has led to the emergence and dissemination of multidrug-resistant isolates. The objective of this study was to assess the rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in calf feces and to characterize their resistance genes for antibiotics like beta-lactams and colistin, but also to determine their virulence genes. Fecal samples were collected from 100 diarrheic calves in the region of Bizerte, Tunisia. After isolation, E. coli isolates were screened for antimicrobial resistance against 21 antibiotics by the disc diffusion method. Characterization of ß-lactamase genes and determination of associated resistance genes were performed by polymerase chain reaction. Among 71 E. coli isolates, 26 (36.6%) strains were ESBL-producing. Most of these isolates were multidrug-resistant (92.3%) and the most prevalent beta-lactamase genes detected were blaCTX-M (n = 26), blaSHV (n = 11), and blaTEM (n = 8), whereas only 1 isolate carried the blaCMY gene. In addition, resistance to carbapenems was detected in two isolates; one of them harbored both blaOXA-48 and blaIMP genes and the other isolate carried only the blaIMP gene. Several resistance genes were identified for the first time in Tunisia from cases of diarrheic calves. Furthermore, to the best of our knowledge, this is the first report of detection and identification of carbapenem resistance genes and virulence genes from calves in North Africa. A high occurrence of antimicrobial resistance of E. coli recovered from fecal samples of calves with diarrhea was observed, highlighting the need for prudent use of antimicrobial agents in veterinary medicine to decrease the incidence of multidrug-resistant bacteria for both animals and humans.

First Report of Extended-Spectrum ß-Lactamase (blaCTX-M1) and Colistin Resistance Gene mcr-1 in E. coli of Lineage ST648 from Cockroaches in Tunisia.

The emergence of multidrug-resistant bacteria has become a major problem. Cockroaches may play an important role in the spread of those bacteria between the environment and humans. This study was designed to screen extended-spectrum ß-lactamase (ESBL)-producing and colistin-resistant strains and to investigate the molecular support of multidrug-resistant Enterobacteriaceae in the external surface and gut homogenates of cockroaches collected from different locations in Tunisia. Between July 2017 and June 2018, 144 Enterobacteriaceae samples were isolated from 115 trapped cockroaches (collective catering, houses, and a hospital). Antibiotic susceptibility testing was performed using the disk diffusion method. Extended-spectrum ß-lactamase-encoding genes and the mcr-1 gene were investigated by real-time PCR (RT-PCR) and standard PCR. The genetic relationship among isolates was studied with the help of multilocus sequence type (MLST) analysis. Of the 144 Enterobacteriaceae isolates, 22 strains exhibited a positive ESBL-screening test (73.3%), including 17 Escherichia coli isolates and 5 Klebsiella pneumoniae isolates. Among them, 9 Escherichia coli isolates were resistant to colistin, with an MIC ranging from 8 to16 µg/L, all of which harbored the mcr-1 gene. Eight blaCTX-M-15 genes were detected; two among them were associated with blaTEM-117 and blaTEM-128, and seven blaCTX-M-1 genes were detected that also harbored the mcr-1 gene. Genotyping analysis revealed 7 different sequence types already described in humans and animals. We report the first survey of mcr-1 in ESBL-producing E. coli isolates from cockroaches. Our findings highlight cockroaches as a source of nosocomial infections, and they are a reservoir of colistin-resistant E. coli, which is a carrier of other additional risk genes such as blaESBL, especially in hospitals. IMPORTANCE Multidrug resistance in Enterobacteriaceae has become a major concern worldwide that is increasingly observed in human, animals, and also cockroaches. In our study, we found that cockroaches may play an important role as a potential vector of multidrug-resistant Enterobacteriaceae in the hospital environment and collective catering. Our study describes the first survey of mcr-1 in ESBL-producing E. coli isolates from hospital cockroaches. Our results further highlight the possibility that mcr-1 may enter humans via cockroach contamination and thereby threaten public health. Our results show that these cockroaches are an important reservoir of colistin-resistant E. coli and carriers of other additional risk genes such as blaESBL, hence the importance of strengthening prevention strategies and of strictly respecting hygiene measures in order to control their distribution and spread in Tunisia.

Prevalence of Escherichia coli O157:H7 isolated from fecal samples of diarrheic camels in Tunisia.

Shiga­toxin­producing E. coli (STEC) is a foodborne pathogen associated with outbreaks worldwide that can be identified in the feces and in the meat of food­producing animals. Our study aimed to evaluate the incidence of E. coli O157:H7 in the feces of diarrheic camels (Camelus dromedarius) in Tunisia. From January 2018 to April 2019, 120 unduplicated fecal samples were obtained from diarrheic camels located in southern Tunisia. Non­sorbitol­fermenting colonies were confirmed as E. coli O157 via latex agglutination test and were screened for the presence of rfbEO157, fliCH7, stx1, stx2, eaeA, and ehxA genes by PCR. All isolates were examined for their susceptibility to 21 antibiotics. Of the 70 E. coli isolates that were recovered from 120 diarrheic camels, 4 (5.7%) were identified as STEC O157:H7. All isolates harbored ehxA and eae genes. Shiga toxin genes stx2 and stx1 were present in 50% and 25% of isolates, respectively. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole­trimethoprim. All isolates belonged to the phylogroup E. This is the first report of E. coli O157:H7 isolates from diarrheic camels in Tunisia with a prevalence of 4 isolates (3.3%) amongst 120 fecal samples. This study supports the necessity for a platform purposed for regular screening and surveillance programs in food­producing animals and meat products, to perform early and rapid identification of food­borne pathogens.

Role of association of OmpK35 and OmpK36 alteration and blaESBL and/or blaAmpC genes in conferring carbapenem resistance among non-carbapenemase-producing Klebsiella pneumoniae.

In Klebsiella pneumoniae, loss of the two major outer membrane porins (OMPs) OmpK35 and OmpK36 confers resistance to carbapenems in strains producing extended-spectrum ß-lactamases (ESBLs) or plasmid-mediated AmpC-type ß-lactamases. This study investigated mechanisms responsible for carbapenem resistance in non-carbapenemase-producing K. pneumoniae (NCPK). All carbapenem-resistant Enterobacteriaceae (CRE) at Charles Nicolle Hospital (Tunis, Tunisia) were collected over a 6-year period (2010-2015). Among the 334 CRE strains collected, 44 (13.2%) were NCPK. MIC ranges for ertapenem, imipenem and meropenem were 1 to >32 mg/L, 0.125-8 mg/L and 0.125-32 mg/L, respectively. All strains showed a multidrug-resistant (MDR) phenotype and were negative for carbapenemase activity. None of the carbapenemase genes searched for were found. ESBL production was confirmed in all isolates except one [CTX-M-15 (n = 39) and SHV-5 (n = 4)]. Three isolates produce DHA-1 (associated with CTX-M-15 in two strains). Molecular fingerprints grouped the 44 NCPK isolates into seven clusters. In seven representative strains of these clusters, SDS-PAGE results showed that four isolates lacked the OmpK35 porin, one isolate lacked OmpK36 and two isolates lacked both OmpK35 and OmpK36. Sequencing of the corresponding porin genes showed amino acid insertions and deletions leading to early termination of translation, point mutations in the promoter region, or insertion sequences disrupting the gene coding sequence. Loss or deficiency of OMPs, coupled with ESBL and/or AmpC production, plays an important role in conferring carbapenem resistance in K. pneumoniae. Dissemination of these MDR bacteria in our hospital may create serious therapeutic problems in the future.