High Prevalence of GES-5 Variant and Co-Expression of VIM-2 and GES-45 among Clinical Pseudomonas aeruginosa Strains in Tunisia.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are a global health concern. The antimicrobial resistance, virulence, and molecular typing of 57 CRPA isolated from 43 patients who attended a specific Tunisian hospital from September 2018 to July 2019 were analyzed. All but one were multidrug-resistant CRPA, and 77% were difficult-to-treat-resistant (DTR) isolates. The blaVIM-2 gene was detected in four strains (6.9%), and among the 36 blaGES-positive CRPA (62%), the blaGES-5 gene was the predominant variant (86%). Three strains co-harbored the blaVIM-2 and blaGES-45 genes, and seven CRPA carried the blaSHV-2a gene (14%). OprD alterations, including truncations by insertion sequences, were observed in 18 strains. Regarding the 46 class 1 integron-positive CRPA (81%), the blaGES-5 gene was located in integron In717, while the blaGES-29 and blaGES-45 genes were found in two new integrons (In2122 and In4879), and the blaVIM-2 gene was found in In1183 and the new integron In2142. Twenty-four PFGE patterns and thirteen sequence types (three new ones) were identified. The predominant serotype O:11 and exoU (81%) were mostly associated with ST235 and the new ST3385 clones. The seven blaSHV-2a-CRPA from different patients belonged to ST3385 and the same PFGE pattern. The blaGES-5- and blaVIM-2 + blaGES-45-positive CRPA recovered mostly from ICU patients belonged to the high-risk clone ST235. Our results highlight the alarming prevalence of blaGES-5- and ST235-CRPA, the co-existence of blaGES-45 and blaVIM-2, and their location within integrons favoring their dissemination.

Serotype Occurrence, Virulence Profiles, Antimicrobial Resistance and Molecular Characterization of Salmonella Isolated from Hospitalized Patients with Gastroenteritis in Great Tunisia between 2010 and 2020.

Non-typhoid Salmonella is one of the major causes of food-borne infections worldwide. The aim of the current study is to determine the serotype occurrence, virulence factors and antimicrobial resistance patterns of Salmonella isolated from hospitalized patients. The identification of Salmonella strains was performed according to REMIC, 2018. The susceptibility of Salmonella isolates was assessed against 20 antimicrobials using the disk diffusion method. Some virulence and antimicrobial resistance genes were identified using PCR. Among the 61 isolated Salmonella strains, seven serotypes were identified and all were positive for the virulence genes invA, mgtC and sirA. Critical resistance rates (>40%) were detected for tetracycline, nalidixic acid, amoxicillin and fluoroquinolones. However, resistances to ertapenem, ceftazidim, aztreonam and colistin were null. In addition, 33% of the isolated strains were multidrug-resistant (MDR). Moreover, 80% and 60% of S. Kentucky isolates were identified as fluoroquinolone-resistant and MDR strains, respectively. The qnrB gene was amplified in 63.2% of fluoroquinolone-resistant strains. The dfrA1 gene was identified in 20% (4/20) of the trimethoprim-sulfamethoxazole resistant strains and the integrase Class 2 gene was amplified in only 8.2% (5/61) of the isolates. Our findings highlight the emergence of MDR Salmonella isolates. A rationalization of antimicrobial use is urgently recommended in both human and veterinary medicine.

First Report of NDM and VIM Coproducing Klebsiella pneumoniae in Tunisia and Emergence of Novel Clones.

Metallo-ß-lactamase (MBL) producing bacteria constitute nowadays a serious global concern worldwide. The purpose of our present study was to characterize molecular features of MBL producing bacteria and to identify the existing clones in our area. Thirteen MBL-producing-Klebsiella pneumoniae were detected in clinical samples from patients hospitalized in the Military hospital of Tunisia during 2017. The molecular research by polymerase chain reaction and sequencing of gene encoding MBL enzymes showed that only two types were identified in our study: blaNDM-1 and blaVIM-1 genes detected, respectively, in eight and six isolates. An association between these two MBL genes (blaNDM-1+blaVIM-1) has been observed in one of our isolates. Other ß-lactamase types (CTXM-15/4 isolates; SHV/2 isolates; OXA-48/3 isolates) were detected in association with New Delhi metallo-beta-lactamase (NDM) and/or Verona Integron-Mediated Metallo-ß-lactamase (VIM) enzymes. Furthermore, these isolates were resistant to other antimicrobial agents, including gentamicin [aac(3)-II/11 isolates], tetracycline (tetB or tetA/2 isolates), chloramphenicol (cmlA and/or floR/3 isolates), streptomycin (aadA/5 isolates), and sulfonamides (sul1 or sul2 or sul3/4isolates). The Multilocus Sequence Typing revealed 10 different Sequence types (ST) of which 7 novel ST: ST147 (3 isolates), ST101 (1 isolate), ST630 (1 isolate), ST3485 (1 isolate), ST3486 (1 isolate), ST3487 (1 isolate), ST3488 (1 isolate), ST3489 (1 isolate), ST3490 (1 isolate), ST3491 (2 isolates). Our study provides new data about MBL producing K. pneumoniae in Tunisia. Thus, we report for the first time the coexpression of blaNDM-1 and blaVIM-1 in our country and also we describe seven novel ST of MBL producing K. pneumoniae in the world.

Huge Diversity of TEM and SHV ß-Lactamases Types Among CTX-M-15-Producing Enterobacteriaceae Species in Tunisia.

The aim of our study was to characterize third-generation cephalosporin (3GC)-resistant Enterobacteriaceae isolated over two different periods from patients hospitalized in the Military Hospital of Tunis with special focus to class A ß-lactamases. This study included 180 Enterobacteriaceae resistant to 3GC isolated from samples of patients hospitalized in various services of the hospital. Enterobacteriaceae species detected by the Vitek 2 Compact® (BioMérieux®) automated system showed the dominance of Klebsiella pneumoniae followed by Escherichia coli during both periods. These strains were mainly isolated from urine samples and rectal swabs of patients hospitalized mostly in neonatology service and intensive care unit. The molecular research of genes encoding CTX-M, TEM, and SHV ß-lactamase types showed a high rate of strains producing CTX-M ß-lactamases, all of them harbored the blaCTX-M-15 gene. However, a huge diversity of SHV and TEM ß-lactamases types was discovered in our study. In fact, nine various subvariants of blaSHV gene (blaSHV-1, blaSHV-11, blaSHV-12, blaSHV-27, blaSHV-28, blaSHV-31, blaSHV-38, blaSHV-79, and blaSHV-81) and eight subvariants of blaTEM gene (blaTEM-70, blaTEM-71, blaTEM-76, blaTEM-77, blaTEM-79, blaTEM-105, blaTEM-148, and blaTEM-186) were identified among our Enterobacteriaceae species during both periods. All subvariants of blaTEM gene and some subvariants of blaSHV gene (blaSHV-31, blaSHV-38, blaSHV-79, and blaSHV-81) have not been previously detected in our country.

Screening for Carbapenemases in Ertapenem-Resistant Enterobacteriaceae Collected at a Tunisian Hospital Between 2014 and 2018.

BACKGROUND: Carbapenem-resistance is frequently detected in Enterobacteriaceae isolated from patients in Tunisia. The study was performed to identify frequent carbapenemases in Tunisian isolates. METHODS: Between May 2014 and January 2018, 197 ertapenem-resistant Enterobacteriaceae were isolated at the microbiological department of the Military Hospital of Tunis. The strains were phenotypically characterized and then subjected to in-house polymerase chain reaction (PCR) targeting the carbapenemase genes blaIMP, blaVIM, blaNDM, blaSPM, blaAIM, blaDIM,blaGIM, blaSIM, blaKPC, blaBIC , and blaOXA-48. RESULTS: The assessed 197 ertapenem-resistant Enterobacteriaceae from Tunis comprised 170 Klebsiella pneumoniae, 19 Enterobacter cloacae, 6 Escherichia coli, 1 Citrobacter sedlakii, and 1 Enterobacter asburiae. Thereby, 55 out of 197 isolates (27.9%) were from blood cultures, suggesting a systemic disease. The carbapenemase gene blaOXA-48 quantitatively dominated by far with 153 detections, followed by blaNDM with 14 detections, which were distributed about the whole study interval. In contrast, blaBIC and blaVIM were only infrequently identified in 5 and 3 cases, respectively, while the other carbapenamases were not observed. CONCLUSIONS: The carbapenemase gene blaOXA-48 was identified in the vast majority of ertapenem-resistant Tunisian Enterobacteriaceae while all other assessed carbapenemases were much less abundant. In a quantitatively relevant minority of isolates, the applied PCR-based screening approach did not identify any carbapenemases.

Vancomycin-Resistant Enterococcus faecium in Tunisia: Emergence of Novel Clones.

Vancomycin-resistant Enterococci (VRE) are a major public health problem worldwide, since they are commonly implicated in nosocomial infections in various regions in the world. The aim of our study was to investigate genetic features and clonal relationship of VRE in the Military hospital of Tunisia. A total of 10 VRE strains were initially detected and identified by the Viteck II compact® (BioMérieux®) automated system, then confirmed by PCR using specific primers. These VRE strains were isolated during the period extended between September 2015 and January 2017 from anal and blood samples from patients hospitalized mainly in the neonatology service and intensive care unit. All these strains were identified as Enterococcus faecium and carried the vanA gene. Other acquired resistance genes were also detected by PCR: [ermB (n = 6); tetL (n = 6); tetM (n = 2); aac(6')-Ie-aph(2'')-Ia (n = 10); aph(3')-III-a (n = 9); ant(6)-Ia (n = 8)]. The insertion sequence IS16 was detected in all our tested strains. Esp virulence gene was detected in only one strain. The clonal relatedness of VRE strains screened by pulse-field gel electrophoresis and multi-locus sequence typing showed four clones: two related clones A1 (one strain) and A2 (one strain) ascribed to ST80 belonged to CC17, the other remaining two clones, named B (one strain) and C (seven strains), revealed two new sequences types assigned to ST1463 and ST1464 respectively. The emergence of novel clones of VRE in this hospital could be a warning of rapid evolution of these resistant bacteria, which calls for new surveillance strategies, strict hygiene and practices.

Occurrence and Characterization of Carbapenemase-Producing Enterobacteriaceae in a Tunisian Hospital.

Carbapenem-resistant Enterobacteriaceae have become of particular concern, since they were quickly disseminated in various areas in the world. The aim of the study was to investigate the prevalence of carbapenemase production among clinical isolates of Enterobacteriaceae recovered from the Military Hospital of Tunisia. Bacterial isolates (n = 125) were recovered from patients in diverse services from March 2014 to February 2016 and identified by Vitek II Compact®. The multiplex PCR for blaVIM, blaIMP, blaNDM, blaKPC, and blaOXA-48 with subsequent amplicon detection by reverse hybridization was performed with the Hyplex SuperBug ID test system (AmplexDiagnostics GmbH, Gars-Bahnhof, Germany). The 125 strains showed resistance to carbapenems of which 102 strains (81.6%) were carbapenemase-producing Enterobacteriaceae and were identified as Klebsiella pneumoniae (85.2%), Enterobacter cloacae (9.8%), Escherichia coli (2.9%), Providencia stuartii (0.9%) and Enterobacter aerogenes (0.9%). These strains were isolated mainly from blood, anal, and urine samples. Patients were mainly hospitalized in the intensive care units, surgery, and medical services. All strains were resistant to ertapenem (100%) and 55.8% showed resistance to imipenem. Carbapenemases genes detected in our study were as follows: blaOXA-48 (84 isolates), blaNDM-1 (8 isolates), blaOXA-48 + blaVIM (5 isolates), and blaOXA-48 + blaNDM-1 (5 isolates). Our research provides epidemiological data showing the quick spread of carbapenem-resistant bacteria in our region, which calls for new surveillance strategies and strict hygiene rules.

First report of nosocomial infection caused by Klebsiella pneumoniae ST147 producing OXA-48 and VEB-8 ß-lactamases in Tunisia.

The aim of this study was to determine the origin of virulence and multiresistance of a Klebsiella pneumoniae isolate from an abdominal wound infection of a patient with a gunshot injury in the thoracoabdominal region. The isolate was identified using biochemical tests and Phoenix™ automated system and was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). MICs of each antibiotic were determined by Etest. Screening for carbapenemase production was performed by the modified Hodge test and was confirmed by PCR amplification. Virulence factors were also studied. Plasmid replicon typing was used to classify Incompatibility (Inc) plasmids harbouring the resistance genes. The transferability of each plasmid was determined by conjugation using Escherichia coli J53. Finally, multilocus sequence typing (MLST) was performed to determine the ST of the strain. The bacterial isolate was identified as K. pneumoniae and was named KPM2, carrying entB, ybtS, mrkD and ycfM virulence genes, but it did not overexpress OqxAB. Isolate KPM2 belonged to ST147 and was classified as resistant to all of the tested antibiotics with MICs above the clinical breakpoints. These resistances were due to production of OXA-48, CMY-2, TEM-1, CTX-M-15 and VEB-8 ß-lactamases. Genetic and molecular studies showed that blaOXA-48 was embedded in transposon Tn1999.2 and was carried by a conjugative IncL/M plasmid of ca. 60kb; blaVEB-8 was harboured on a conjugative IncA/C plasmid of ca. 120kb. This study confirmed that the resistance conferred by OXA-48 and VEB-8 contributed to the failure of antibiotic treatment and consequently death of the patient.

Clonal lineages detected amongst tetracycline-resistant meticillin-resistant Staphylococcus aureus isolates of a Tunisian hospital, with detection of lineage ST398.

Tetracycline resistance has been postulated as a potential phenotypic marker of livestock-associated lineage ST398 amongst meticillin-resistant Staphylococcus aureus (MRSA) clinical isolates in some European hospitals. The objective of this study was to determine if this marker could also be applied to Maghrebian countries. In total, 99 MRSA isolates were collected in a Tunisian hospital during January 2011-October 2012, and 24 tetracycline-resistant MRSA isolates of this collection were characterized. All isolates were tested for antimicrobial resistance phenotypes and genotypes, molecular typing, and virulence genes. Multilocus sequence typing showed that the majority of the isolates (19/24) belonged to clonal complex CC8 (ST247, n = 12 isolates; ST239, n = 6 isolates; ST241, n = 1 isolate). The remaining isolates belonged to CC398 (ST398, n = 1 isolate), CC5 (ST5 and ST641, n = 2 isolates), and CC80 (ST728, n = 2 isolates). Spa typing discriminated MRSA in eight spa types: bib26 (n = 12 isolates), bib26 (n = 5 isolates), bib26 (n = 2 isolates), and bib26, bib26, bib26, bib26 and the new bib26 (n = 1 isolate each). Three agr groups were found amongst the studied isolates: agr group I (n = 20 isolates), agr group II (n = 2) and agr group III (n = 2 isolates). We report the detection of one MRSA ST398-t899 isolate in the nasal sample of a farmer patient in Tunisia, representing the first report of ST398 in humans in Africa. Tetracycline resistance seems not to be a good phenotypic marker for MRSA ST398 strains in Tunisia, where CC8 was the most prevalent lineage. Continuous efforts to understand the changing epidemiology of this micro-organism are necessary not only for appropriate antimicrobial treatment and effective infection control, but also to monitor its evolution.

vanA-containing E. faecium isolates of clonal complex CC17 in clinical and environmental samples in a Tunisian hospital.

Twenty-eight vancomycin (VA)-resistant enterococci isolated from different patients (n = 16) and also from the environment (n = 12) were recovered in a Tunisian military hospital during 2012-2013. The mechanisms of resistance to VA and to other antibiotics as well as the presence of esp and hyl virulence genes were determined in these isolates by PCR, being their clonal relationship analyzed by pulsed-field gel electrophoresis (PFGE). VA resistance mechanisms detected were as follows (species-patient/environment): vanA (Enterococcus faecium, 13/5), vanC1 (Enterococcus gallinarum, 3/0), and vanC2 (Enterococcus casseliflavus, 0/7). Most of the VA-resistant enterococci presented a multiresistance phenotype and harbored different resistance genes (erm(B), tet(M), tet(L), ant(6)-Ia, aac(6')-aph(2"), aph(3')-IIIa, and catA). The PFGE revealed the presence of 3 clones (A, B, C) and 1 closely related pattern (A1) among the 13 vanA-containing E. faecium isolates of patients showing 11 of them the A-A1 patterns. The clone A was also detected in all 5 environmental vanA-containing E. faecium isolates. Strains did not contain esp or hyl virulence genes. Multilocus sequence typing was performed in 4 E. faecium isolates representative of the 4 detected pulsotypes (A, A1, B, and C), and 2 different sequence types were identified (ST18 and ST80), both of them included in clonal complex CC17. These strains contained the IS16 element and showed ampicillin and ciprofloxacin resistance. VA resistance could be an emerging problem in Tunisia, and this is one of the first cases described so far in this country.