High rates of intestinal colonization with carbapenemase producing Enterobacteriaceae in hematopoietic stem cell transplant recipients.

Carbapenem resistant Enterobacteriaceae (CRE) are major human pathogens because, these cause high number of difficult-to-treat infections. Allogeneic hematopoietic stem cell transplant (AHSCT) recipients are highly exposed to these type of bacteria. The aim of our study was to investigate prevalence of CRE colonization in AHSCT patients and to determine genes encoding carbapenem resistance. A retrospective study conducted between January 2015 and December 2019, involved 55 patients colonized with CRE strains. We determined the rate of antibiotic resistance according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the carbapenem resistance genes by PCR assays for genes encoding most frequent ß-lactamases namely, blaGES, blaKPC, blaIMI, blaNDM, blaVIM, blaIMP and blaOXA-48. Eighty-one episodes of CRE colonization were recorded in 55 patients, mainly suffering from acute leukaemia (30%) and aplastic anemia (26%). History of hospitalization was noted in 80 episodes. Prior antibiotic treatment, severe neutropenia and corticosteroid therapy were respectively found in 94%, 76% and 58% of cases. Among the 55 patients, six patients (11%) developed a CRE infection. The CRE responsible for colonization were carbapenemase producers in 90% of cases. They belonged mostly to Klebsiella pneumoniae (61/81) and Escherichia coli species (10/81). Antibiotic resistance rates were 100% for ertapenem, 53% for imipenem, 42% for amikacin, 88% for ciprofloxacin and 27% for fosfomycin. Molecular study showed that blaOXA-48 gene was the most frequent (60.5%), followed by blaNDM (58%). Thirty-five (43%) strains were co-producers of carbapenemases. In our study, we report a high rate of CRE intestinal colonization in AHSCT recipients of our center.

Co-occurrence of blaNDM-1 and blaOXA-23 in carbapenemase-producing Acinetobacter baumannii belonging to high-risk lineages isolated from burn patients in Tunisia.

AIMS: Carbapenem-resistant Acinetobacter baumannii (CR-Ab) is an important cause of infections in burn patients. This study aimed to characterize the antimicrobial susceptibility pattern of CR-Ab isolated from burns in Burn Intensive Care Unit (BICU) of the Trauma and Burn Centre of Ben Arous, to determine the prevalence of ß-lactamase-encoding genes and to search eventual genetic relatedness of CR-Ab strains. METHODS AND RESULTS: From 15 December 2016 to 2 April 2017, all nonduplicated CR-Ab isolated in burn patients in the BICU were screened by simplex Polymerase Chain Reaction (PCR) for the class A, B, C, and D ß-lactamase genes. Sequencing was performed for NDM gene only. Genetic relatedness was determined by using pulsed field gel electrophoresis (PFGE) and by multilocus sequence typing. During the study period, 34 strains of CR-Ab were isolated in burns, mainly in blood culture (n = 14) and central vascular catheter (n = 10). CR-Ab strains were susceptible to colistin but resistant to amikacin (91%), ciprofloxacin (100%), rifampicin (97%), and trimethoprim-sulfamethoxazole (100%). All strains harbored blaOXA-51-like and blaOXA-23 genes, only or associated to blaGES (n = 26; 76%), blaADC (n = 20; 59%), blaPER-1 (n = 6; 18%) or/and blaNDM-1 (n = 3; 9%). PFGE identified 16 different clusters and revealed that most strains belonged to one major cluster A (n = 15; 44.1%). Among NDM-1 isolates, two were clonally related in PFGE and belonged to two single locus variant sequence type ST-6 and ST-85. CONCLUSIONS: This is the first description of clonally related NDM-1 and OXA-23-producing A. baumannii strains in the largest Tunisian BICU associated with two single locus variant sequence types ST6 and ST85.

Outbreak caused by pandrug-resistant blaOXA-69/blaOXA-23/blaGES harboring Acinetobacter baumannii ST2 in an intensive care unit.

Acinetobacter baumannii has emerged as a main nosocomial pathogen exhibiting high rates of resistance to clinically relevant antibiotics. Six pandrug-resistant A. baumannii (PDR-A. baumannii) were recovered from three patients in a Tunisian Intensive Care Unit (ICU) between 10th and 16th of May 2018 resulting in one fatal case and raising the possibility of an outbreak. On 18th of May environmental screening of ICU surfaces was carried out. On 22nd of May a fourth patient was infected with PDR-A. baumannii and died. A second investigation was carried out for environmental screening and PDR-A. baumannii was isolated from the respirator. Antimicrobial susceptibility testing was performed according to EUCAST (2019) guidelines. MIC of colistin was determined by broth microdilution method. PCR was used to detect 14 beta-lactamases/carbapenemases and mcr (mcr-1 to mcr-5) genes. The genetic relatedness of PDR-A. baumannii isolates was determined by PFGE and MLST. Seven PDR-A. baumannii isolates were recovered from four patients, one MDR strain from wash basin, a PDR strain from hand sanitizer bottle and another PDR strain from respirator. All PDR-A. baumannii (n = 9) harbored blaOXA-69 gene and none carried mcr. Moreover, seven carried blaGES and blaOXA-23 genes. PFGE identified four pulsotypes (A, B, C, and D) with the pulsotype A gathering seven PDR-A. baumannii isolates: six from three patients and one from hygiene sample. MLST revealed that all PDR-A. baumannii isolates of pulsotype A belonged to the pandemic clone ST2. Systematic screening of MDR and PDR-A. baumannii is highly recommended to limit dissemination of such strains in ICUs.

Detection of carbapenem resistant Pseudomonas aeruginosa co-harboring blaVIM-2 and blaGES-5 in burn patients.

Pseudomonas aeruginosa is one of the major infectious agents in burn patients. Globally, high rates of antimicrobial resistance in P. aeruginosa have been reported, which is a cause of concern. The objective of this study was to determine the rate of resistance to carbapenems in P. aeruginosa isolates recovered from burn patients in Tunisia, to search genes encoding for carbapenemases and to determine their epidemiological markers (serotypes). A retrospective study was conducted in the Burn Intensive Care Unit (BICU) of the Trauma and Burn Centre of Ben Arous, Tunisia, and P. aeruginosa isolates collected from burn patients, from January to December 2018 were investigated. Carbapenemase screening was performed by Carbapenem Inactivation Method (CIM) and by EDTA-disk test for all carbapenem resistant isolates. Genes encoding carbapenemases (blaVIM, blaIMP, blaGES, blaNDM, and blaKPC) were investigated by PCR and selected carbapenemase genes were sequenced. During the study period, 104 non duplicated P. aeruginosa isolates were recovered. Most of them were isolated from skin samples (45.1%) and blood culture (22.1%) and belonged to O:11 (19.2%), O:12, and O:5 (12.5%, each) serotypes. High rates of resistance were observed for carbapenems (64.4%). Among the 67 carbapenem resistant isolates, 58 (86.5%) harbored blaVIM gene and 55 (82%) blaGES gene; in addition, 48 (71.6%) co-harbored blaVIM and blaGES genes. After sequencing, the blaVIM-2 and blaGES-5 gene variants were identified in seven randomly selected isolates. To the best of our knowledge, this is the first description of P. aeruginosa simultaneously harboring blaVIM-2 and blaGES-5 genes.

Diagnostic low-dose X-ray radiation induces fluoroquinolone resistance in pathogenic bacteria.

PURPOSE: The crisis of antibiotic resistance has been attributed to the overuse or misuse of these medications. However, exposure of bacteria to physical stresses such as X-ray radiation, can also lead to the development of resistance to antibiotics. The present study aimed to investigate the effect of exposure to diagnostic low-dose X-ray radiation on the bacterial response to antibiotics in two pathogenic bacteria, including the Gram-positive Staphylococcus aureus and Gram-negative Salmonella enteritidis. METHODS: The bacterial strains were exposed to diagnostic X-ray doses of 5 and 10 mGy, which are equivalent to the doses delivered to patients during conventional radiography X-ray examinations in accordance with the European guidelines on quality criteria for diagnostic radiographic images. Following exposure to X-ray radiation, the samples were used to estimate bacterial growth dynamics and perform antibiotic susceptibility tests. RESULTS: The results indicate that exposure to diagnostic low-dose X-ray radiation increased the number of viable bacterial colonies of both Staphylococcus aureus and Salmonella enteritidis and caused a significant change in bacterial susceptibility to antibiotics. For instance, in Staphylococcus aureus, the diameter of the inhibition zones for marbofloxacin decreased from 29.66 mm before irradiation to 7 mm after irradiation. A significant decrease in the inhibition zone was also observed for penicillin. In the case of Salmonella enteritidis, the diameter of the inhibition zone for marbofloxacin was 29 mm in unexposed bacteria but decreased to 15.66 mm after exposure to 10 mGy of X-ray radiation. Furthermore, a significant decrease in the inhibition zone was detected for amoxicillin and amoxicillin/clavulanic acid (AMC). CONCLUSION: It is concluded that exposure to diagnostic X-ray radiation can significantly alter bacterial susceptibility to antibiotics. This irradiation decreased the effectiveness of fluoroquinolone and ß-lactam antibiotics. Specifically, low-dose X-rays made Staphylococcus aureus resistant to marbofloxacin and increased its resistance to penicillin. Similarly, Salmonella Enteritidis became resistant to both marbofloxacin and enrofloxacin, and showed reduced sensitivity to amoxicillin and AMC.

Occurrence of High-Risk Clonal Lineages ST58, ST69, ST224, and ST410 among Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated from Healthy Free-Range Chickens (Gallus gallus domesticus) in a Rural Region in Tunisia.

Antimicrobial-resistant Escherichia coli isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing E. coli (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 blaCTX-M-1, 5 blaCTX-M-55, 5 blaCTX-M-15, 1 blaSHV-2, and 1 blaSHV-12. Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by aac(6')-Ib-cr (n = 21), qnrB (n = 1), and qnrS (n = 2); tetA (n = 17)/tetB (n = 26); sul1 (n = 29)/sul2 (n = 18); and mcr-2 (n = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic E. coli. Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes fyuA, fimH, papGIII, and iutA were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones.

Dissemination of epidemic ST239/ST241-t037-agrI-SCCmecIII methicillin-resistant Staphylococcus aureus in a Tunisian trauma burn intensive care unit.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing health care-infections in the world, especially in burns. The aim of this study was to assess the extent of dissemination of MRSA isolated from burn patients in Burn Intensive Care Unit in Tunisia and to evaluate the frequency of virulence and antibiotics resistance genes. Among the 72 S. aureus isolates analyzed in the study, 54% were MRSA. The majority of MRSA (94.8%) were multidrug resistant and they had a high resistance rates to kanamycin (94.8%), tobramycin (90%), tetracycline (94.8%) and ciprofloxacin and rifampicin (87%, each). The gene aac(6')-Ie-aph(2″)-Ia conferring resistance to kanamycine and tobtamycin were detected in all isolates and the aph(3')-Ia gene conferring resistance to gentamicin were detected in 2.8% of resistant isolates. Tetracycline resistance genes tet(M), tet(K) and tet(L) were detected in 100%, 10.8% and 2.8% of the isolates, respectively. The SCCmec type III and the agr type I were the most predominant (69.2% and 90%, respectively). The 27 SCCmecIII-agrI isolates were clustered into two PFGE types A and B. The two representative isolates of PFGE clusters A and B belonged to ST239-t037 and ST241-t037 respectively. As conclusion, our results showed a high prevalence of MRSA in trauma burn intensive care unit belonging to two multidrug resistant clones ST239/ST241-agrI-t037-SCCmecIII MRSA. We also demonstrated that MRSA was disseminated between burn patients.

An intermittent outbreak of Burkholderia cepacia contaminating hematopoietic stem cells resulting in infusate-related blood stream infections.

Microbial contamination of hematopoietic stem cells (HSC), used for autologous and allogenic transplantations, is rare but could cause serious blood stream infection in transplanted patients. These infections occur immediately, or later following the formation of biofilm on the catheter lumen. The present study describes an intermittent B. cepacia HSC contamination associated with nosocomial bacteremia: from October 2011 to April 2015, 17 B. cepacia strains were isolated in HSC bags (n = 14) and blood cultures (n = 3) in patients hospitalized in the National Bone Marrow Transplant Center. Two epidemiologic investigations in the National Blood Transfusion Center, allowing the isolation of three strains in hygiene samples, and four interventions in this institution were done. To identify the source of this contamination, a molecular investigation was done on 23 B. cepacia strains isolated in our center from 2007 to 2015. PFGE analysis revealed five clusters. The major cluster included 18 strains isolated from HSC bags (n = 14), blood culture (n = 1), and water cans and bath (n = 3). The second cluster (B) including only two and the remaining clusters (C, D, and E) contained single strains isolated before the epidemic period. These findings confirmed that the origin of the outbreak was the contaminated water used in the water bath during the thawing step of HSC bags. Based on this result, new sterile water was used for every defrosting, but HSC bags contamination persisted. In May 2015, the water bath was replaced with a dry bath and no B. cepacia strain was isolated from that date to April 2020.

Species distribution and genes encoding antimicrobial resistance in enterococcus spp. isolates from rabbits residing in diverse ecosystems: A new reservoir of linezolid and vancomycin resistance.

AIMS: Worldwide, studies regarding antimicrobial resistance in rabbits are scarce. In addition, it seems that rearing conditions have important impact on emergence and spread of antimicrobial-resistant bacteria. Thus, the authors sought to (1) assess the role of rabbits residing across diverse ecosystems as potential reservoirs of antimicrobial-resistant enterococci and (2) investigate the genetic background of detected resistances. METHODS AND RESULTS: Faecal samples from 60 healthy farmed rabbits (one farm), 35 laboratory rabbits and 31 wild rabbits were analysed. Overall, 97 enterococci isolates were accumulated, as follows: 44 E. faecium, 37 E. faecalis, 7 E. gallinarum, 5 E. durans and 4 E. avium. E. faecalis isolates were statistically associated with farm rabbits and wild rabbits (p < 0.05). High rates of resistance were observed for tetracycline (60.8%; tetM [n = 48; 81.3%], tetO [n = 7; 11.8%] and tetL [n = 1; 1.7%]), erythromycin (43.3%; msr(A) [n = 14; 33.3%] and ermB [n = 13; 31%]), ampicillin (29.9%), streptomycin (26.8%; ant(6)-Ia [n = 3, 11.5%]) and vancomycin (21.6%; vanA [one E. faecium + one E. faecalis; 9.5%]). Low frequencies of resistance were observed for teicoplanin (9.2%), linezolid (8.2%), ciprofloxacin (7.2%) and gentamicin (1%; aac(6')-Ie-aph(2″)-Ia). Resistance to ampicillin and vancomycin was associated with laboratory rabbits (p < 0.05). Int-Tn (Tn916/1545) was detected in 27 (27.8%) isolates, of which 10 isolates co-harboured tetM and ermB genes, while 16 comprised tetM. CONCLUSION: Findings indicate that clinically relevant enterococci species isolated from rabbits are frequently resistant to antimicrobials and harbour a range of genes associated with the Tn916/1545 family. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the high rates of antimicrobial-resistant enterococci from rabbits and the occurrence of both vancomycin- and linezolid-resistant isolates, potentially representing a very serious threat to human and animal health.

Genetic characterization of ESBL/pAmpC-producing Escherichia coli isolated from forest, urban park and cereal culture soils.

Little is known about the role of forestland and non-fertilized agriculture soils as reservoirs of extended-spectrum beta-lactamase (ESBL) and plasmid-borne AmpC (pAmpC)-producing Escherichia coli isolates. Thus, in the present study, 210 soil samples from various origins (forest of Oued Zen (Ain Drahem), non-agriculture soils from different park gardens in Tunis City, cereal culture soils and home gardens) were investigated to characterize cefotaxime-resistant E. coli isolates. A total of 22 ESBL/pAmpC-producing E. coli were collected, and all harbored variants of the blaCTX-M gene (15 blaCTX-M-1, 5 blaCTX-M-55 and 2 blaCTX-M-15). A total of seven and two isolates harbored also blaEBC and blaDHA-like genes, respectively. Resistances to tetracycline, sulfonamides and fluoroquinolones were encoded by tetA (n = 4)/tetB (n = 12), sul1 (n = 17)/sul2 (n = 19) and aac(6')-Ib-cr (n = 2)/qnrA (n = 1)/qnrS (n = 1) genes, respectively. A total of seven isolates were able to transfer by conjugation cefotaxime-resistance in association or not with other resistance markers. PFGE showed that ten and two isolates were clonally related (pulsotypes P1 and P2). The 10 P1 isolates had been collected from forestland, cereal culture soils and an urban park garden in Tunis City, arguing for a large spread of clonal strains. Our findings highlight the occurrence of ESBL/pAmpC-E. coli isolates in soils under limited anthropogenic activities and the predominance of CTX-M enzymes that are largely disseminated in E. coli from humans and animals in Tunisia.

Coagulase negative Staphylococcus bacteremia in hematopoietic stem cell transplant recipients: Clinical features and molecular characterization.

The purpose of our study was to investigate the epidemiology of coagulase negative staphylococci (CoNS) responsible for bacteremia in hematopoietic stem cell transplant (HSCT) recipients and to determine the prevalence and the genetic background of methicillin resistance. The prevalence of CoNS bacteremia was 7.4% (54/728), higher in allograft (10.7%) than in autograft (4.7%) recipients. A sepsis or a septic shock were observed in 9% of cases. No deaths were attributable to CoNS bacteremia. The methicillin resistance rate was 81%. All MR-CoNS, harbored mecA gene and 90% were typeable with SCCmec typing using PCR amplification. The SCCmec type IV was the most frequent (44%). Clonal dissemination of MR- Staphylococcus epidermidis strains was limited. Our study showed a low prevalence and favorable outcome of CoNS bacteremia in HSCT recipients with limited clonal diffusion. However, they were associated with a significant rate of severe infections and a high rate of methicillin resistance, mediated by SCCmec IV element in most cases.

Multidrug resistant bacteremia in hematopoietic stem cell transplant recipients.

BACKGROUND: Bacteremia become fearsome in hematopoietic stem cell transplant (HSCT) recipients with the emergence of multidrug-resistant (MDR) strains. AIM: Our purpose was to investigate the prevalence of MDR bacteremia in HSCT recipients at the Tunisian National Bone Marrow Transplant Center, associated factors and attributable mortality rate. METHODS: Our retrospective study (January 2010-December 2017) included all MDR bacteremia in the Hematology department. MDR rods were: extended spectrum beta-lactamase producing Enterobacterales (ESBL-E), P. aeruginosa and A. baumannii resistant to at least three families of antibiotics, methicillin-resistant S. aureus (MRSA) and vancomycin resistant E. faecium (VRE). RESULTS: The prevalence of MDR bacteremia among HSCT recipients was 5.9% (48/816) with a stable trend over time (rs=0.18). Neutropenia, prior hospitalization, prior antibiotherapy and prior colonization with MDR pathogens were observed in 59%, 58%, 48% and 31% of cases, respectively. Imipenem was the most prescribed first-line antibiotic (50%). The attributable mortality rate was 13%. MDR bacteria (n=48) belonged to ESBL-E (60%), P. aeruginosa (19%), A. baumannii (13%), MRSA (4%) and VRE (4%). For ESBL-E and P. aeruginosa, the rates of antibiotic resistance were respectively, 17% and 44% to imipenem, 31% and 56% to amikacin and 15% and 0% to colistin. Strains of A. baumannii were susceptible only to colistin. The MRSA (n=2) were resistant to ciprofloxacin and gentamicin and susceptible to glycopeptides. The VRE (n=2) were susceptible to linezolid and tigecycline. CONCLUSION: Low prevalence of MDR bacteremia in HSCT recipients but high attributable mortality rate, requiring reinforcement of hygiene measures.

Clonal spread of PER-1 and OXA-23 producing extensively drug resistant Acinetobacter baumannii during an outbreak in a burn intensive care unit in Tunisia.

Extensively drug resistant Acinetobacter baumannii (XDR-Ab), has emerged as an important pathogen in several outbreaks. The aim of our study was to investigate the eventual genetic relatedness of XDR-Ab strains recovered from burn patients and environment sites in the largest Tunisian Burn Intensive Care Unit (BICU) and to characterize ß-lactamase encoding genes in these strains. Between March 04th, 2019 and April 22nd, 2019 an outbreak of XDR-Ab was suspected. Environmental screening was done. All isolates were screened by simplex PCR for ß-lactamase genes. Genetic relatedness was determined by pulsed field gel electrophoresis (PFGE) of ApaI-digested total DNA. During the study period, 21 strains of A. baumannii were isolated in burn patients, mainly in blood culture (n = 7) and central vascular catheter (n = 6). All strains were susceptible to colistin but resistant to imipenem (n = 23), ciprofloxacin (n = 23), amikacin (n = 22), tigecyclin (n = 5) and rifampicin (n = 4). The blaOXA-51-like, blaOXA23, and blaADC genes were present in all strains. These resistance determinants were associated with blaPER-1 in 10 strains. The ISAba1 was inserted upstream of blaOXA-23 in all isolates. PFGE revealed two major clusters A (n = 11) and B (n = 5). This is the first description in Tunisia of clonally related PER-1 producing XDR-Ab in burn patients with probable environmental origin.

Epidemiology and molecular characterisation of colistin-resistant Klebsiella pneumoniae isolates from immunocompromised patients in Tunisia.

The aim of this retrospective study was to investigate the resistance determinants and genetic relatedness of colistin-resistant Klebsiella pneumoniae (ColR-Kp) isolates in the National Bone Marrow Transplant Center of Tunis (NBMTC) between 2002 and 2013. Minimum inhibitory concentrations (MICs) for colistin of ColR-Kp isolates were assessed using a UMIC broth microdilution kit. All isolates were screened by PCR for the resistance genes mcr-1, blaCTX-M-1, blaSHV, blaTEM, blaOXA-1, blaOXA-48, blaKPC, blaIMP, blaVIM, blaNDM, blaGES, blaIMI and blaAmpC as well as class 1, 2 and 3 integrons. The clonality of ColR-Kp isolates was investigated by pulsed-field gel electrophoresis (PFGE). A total of 24 non-duplicate ColR-Kp isolates were collected with MICs ranging from 16 mg/L to >64 mg/L, representing 3.4% (24/709) of the total K. pneumoniae isolates. The colistin resistance gene mcr-1 was not found. The blaSHV gene was present in all of the isolates, blaTEM in 18 isolates, blaCTX-M-1 and blaOXA-1 in 15 isolates each and blaCMY-16 in two isolates. The blaOXA-48 gene was found in five carbapenem-resistant isolates and was associated with blaCTX-M-1, blaSHV, blaTEM and blaOXA-1. All isolates were negative for the remaining carbapenemases. Class 1 integron was found in 19 isolates. PFGE showed non-clonal spread of ColR-Kp isolates in this center. The increasing rate of colistin resistance in K. pneumoniae isolates in NBMTC was neither associated to clonal diffusion nor to the plasmid-mediated colistin resistance gene mcr-1. Its association with the use of colistin in total digestive decolonization in transplant patients at NBMTC must be investigated.

High prevalence of multidrug-resistant international clones among macrolide-resistant Streptococcus pneumoniae isolates in immunocompromised patients in Tunisia.

OBJECTIVES: Macrolide-resistant Streptococcus pneumoniae isolates have increased considerably in the last decade, with important geographical variations in involved phenotypes and genotypes. The aim of this study was to investigate phenotypes, genotypes, serotypes and genetic relatedness of macrolide-resistant S. pneumoniae isolated from immunocompromised patients in Tunisia. METHODS: Antibiotic susceptibility was determined by disk diffusion, and MICs of erythromycin and clindamycin were determined for macrolide-resistant isolates by Etest. Macrolide-resistant isolates were analysed by PCR for ermB, mefA, tetM, tetO and Int-Tn1545. Serotyping was done by multiplex PCR and the Quellung reaction. Multilocus sequence typing (MLST) was performed for molecular typing. RESULTS: Macrolide resistance was observed in 41 (69.5%) of 59 isolates. Of the 41 isolates, 37 (90.2%) had a macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype, with a predominance of high-level inducible MLSB phenotype, and harboured the ermB gene. All isolates with high-level inducible MLSB phenotype were highly resistant to erythromycin and clindamycin. Four isolates (9.8%) had a macrolide (M) resistance phenotype and harboured the mefA gene. Erythromycin-resistant isolates were multidrug-resistant (MDR) in 97.5% of cases and extensively drug-resistant in 12.2%. The isolates belonged essentially to four serotypes (19F, 23F, 14 and 6B). They were mainly assigned to three sequence types (ST81, ST2918 and ST386). Also, 65.9% of the isolates were grouped in three clonal complexes (CC81, CC838 and CC386). CONCLUSIONS: These data indicate a high prevalence of Tn1545 transposon and of three MDR international clones contributing to the high frequency of multidrug resistance among S. pneumoniae isolates in our centre.

Description of a novel mutation in the atpC gene in optochin-resistant Streptococcus pneumoniae strains isolates from Tunisia.

Identification of Streptococcus pneumoniae among other α-haemolytic streptococci is based on phenotypic or genotypic characteristics such as colony morphology, bile solubility and optochin susceptibility. This study reports three optochin-resistant S. pneumoniae strains isolated from immunocompromised patients in Tunisia. The three isolates were positive for the bile solubility test. Biochemical identification with API® 20 Strep was not discriminatory for two strains. The three strains had different serotypes (6C, 19F and 23F) and three different sequence types (ST386, ST320 and ST326). Sequencing of the atpA and atpC genes for each strain showed only modification in atpC. The mutations Met13→Val or Val48→Ile were observed in two strains. However, in the third strain a novel type of mutation (Val15→Ile) was identified.

Molecular characterisation and epidemiology of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolates from immunocompromised patients in Tunisia.

OBJECTIVES: This study investigated the prevalence of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP) strains in the National Bone Marrow Transplant Center of Tunis between 2002 and 2011 as well as their associated antimicrobial resistance patterns and molecular features. METHODS: Antimicrobial susceptibility was determined by the disk diffusion method according to CA-SFM guidelines. All of the strains were screened for ß-lactamase genes, plasmid-encoded AmpC genes and integrons. Carbapenemase genes were analysed by PCR and sequencing for strains showing reduced susceptibility to ertapenem. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequencing typing (MLST). RESULTS: A total of 128 non-repetitive ESBL-KP strains (23.4%) responsible for infection or colonisation were recovered among 548 K. pneumoniae strains. The isolates were also multidrug-resistant. Molecular analysis revealed the prevalence of blaSHV-type (92.2%), followed by blaOXA-1 (81.3%) and blaCTX-M-1 group (73.4%). Four ertapenem-resistant ESBL-KP strains (3.1%) carried the blaOXA-48 gene associated with the blaCTX-M-15 gene. Class 1 integrons were the most prevalent among the isolates (85.2%). High diversity was demonstrated by PFGE with limited clonal dissemination of 1 major (n=13 strains) and 11 minor clusters (each comprising 2-3 strains). MLST of representative strains also showed high diversity with two main epidemic clones: ST15, associated with the major cluster; and ST101, associated with five minor clusters (n=11 strains). CONCLUSIONS: This study provides relevant information on the epidemiology of ESBL-KP strains in oncohaematology patients, of which 18.8% belonged to the specific CTX-M-15 K. pneumoniae clones ST15 and ST101.

Serotype Distribution, Antibiotic Resistance and Clonality of Streptococcus pneumoniae Isolated from Immunocompromised Patients in Tunisia.

BACKGROUND: Pneumococcal disease, a major cause of morbidity and mortality globally, has higher incidence among young children, the elderly and the immunocompromised of all ages. In Tunisia, pneumococcal conjugate vaccines (PCVs) are not included in the national immunization program. Also, few studies have described the epidemiology of S. pneumoniae in this country and, in particular, no molecular typing studies have been performed. The aim of this study was to evaluate serotype distribution, antimicrobial resistance and clonality of Streptococcus pneumoniae isolated from neutropenic patients in Tunisia. METHODS: Fifty-nine S. pneumoniae were isolated from infection (n = 31) and colonization (n = 28) sites of patients (children and adults) attending the National Centre of Bone Marrow Transplantation in Tunis between 2005-2011. All isolates were characterized by serotype, antimicrobial resistance pattern and multilocus sequence typing (MLST). RESULTS: The majority (66.1%) of the isolates belonged to five serotypes all included in PCVs: 6B, 9V, 14, 19F and 23F. The potential coverage of the 10-valent and 13-valent PCV was of 71.2% and 76.3% respectively. Resistance rates were very high and 69.5% of the isolates were multidrug resistant: non-susceptibility rates to penicillin, amoxicillin and cefotaxime were 66.1%, 40.7% and 27.1%, respectively; resistance rates to erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole, were 69.5%, 61.0%, 37.3%, 22.0% and 67.8%, respectively. The most frequent serotypes had STs characteristic of multidrug resistant international clones known to be highly successful and important causes of pneumococcal infection: Spain 23F-ST81, France 9V/14-ST156, Spain 6B-ST90, 19F-ST320, and Portugal 19F-ST177. CONCLUSIONS: The majority of S. pneumoniae strains recovered from immunocompromised patients in Tunisia are representatives of multidrug resistant pandemic clones that express serotypes targeted by PCVs. To contain the burden of pneumococcal disease and improve treatment choices among Tunisian immunocompromised patients PCVs should be offered to all of them.