HPV16 E2 variants correlated with radiotherapy treatment and biological significance in cervical cell carcinoma.

Specific genetic mutations in human papillomavirus type 16 (HPV16) DNA are considered important in cervical lesion progression. This study analyzes to what extent radiotherapy treatment contributes to viral DNA mutation in cervical cell carcinomas, and the biological significance of these mutations. Serial tumor tissue, including 44 cervical cancer samples, collected before and after radiotherapy, and 52 biopsies with benign cervices, were tested and analyzed for the presence of HPV16, and for the integrity of the E2 gene. Analysis was performed with polymerase chain reaction (PCR), and a bidirectional sequencing assay was performed to find HPV16 E2 gene variants. HPV16 E2 accounted for 81.8% and 37.5% among tumor and benign cervices respectively (p = 0.02). The incremental number of DNA mutations was associated with radiotherapy treatment. Most E2 gene mutations involved regions encoding the amino-terminal and carboxy-terminal regions of E2 in the tumor irradiated samples. Amino acid changes T135 K, A143T, N203D and P208A in the amino-terminal region were the most common mutations across the irradiated samples. Rather, the mutations in the carboxy-terminal region (T3694A and T3805G) were synonymous changes. Specific nucleotide deletions were detected in the hinge domain, at positions 3455A > -, 3466 T > -, and 3501A > -. The mutation degree is influenced by the irradiation modalities, interestingly E2 sequence mutation being found widely after radiotherapy treatment with a total fractioned dose of 50 Gy (p = 0.004). E2 mutation has predictive and biological significance in cervical cancer patients receiving curative radiation therapy. Possibly, E2 mutation could influence viral genome intactness and could serve as an intrinsic marker for cervical cancer.

An Outbreak of NDM-1-Producing Klebsiella pneumoniae, Associated with OmpK35 and OmpK36 Porin Loss in Tunisia.

OBJECTIVES: To describe clinical and molecular characteristics of an outbreak due to metallo-ß-lactamases (MBLs) producing Klebsiella pneumoniae collected at Charles Nicolle Hospital of Tunis and to analyze the impact of outer membrane porin (OMP) loss on carbapenem resistance levels. METHODS: Between 2010 and 2015, 178 carbapenem-resistant Enterobacteriaceae were isolated. Screening for MBL production was performed using combined disk diffusion method, with imipenem and ethylene diamine tetraacetic acid (EDTA) as inhibitors. Resistance genes and virulence factors were identified by polymerase chain reaction (PCR) and sequencing. Genotyping was performed by pulsed-field gel electrophoresis and multilocus sequence typing. Genetic environment of carbapenemase genes was determined by PCR mapping. Conjugation assays were performed, and plasmids were assigned to incompatibility groups by PCR-based replicon typing. OMPs were profiled by sodium dodecyl sulfate-polyacrilamide gel electrophoresis, and porin genes were sequenced. RESULTS: Nineteen K. pneumoniae (10.6%) showing MBL activity were isolated from patients hospitalized on four different wards. NDM-1 was the only MBL identified, in association with blaOXA-48. All strains lacked at least one OMP, and carbapenem resistance levels were remarkably elevated in strains lacking OmpK35 and OmpK36. blaNDM-1 was located in IncFIA-type conjugative plasmid, with the same genetic context in all strains. The epidemiological diffusion of blaNDM-1 was due to two clones, one major clone belonging to sequence type (ST) 147 (n = 16) and the other clone belonging to ST307 (n = 3). CONCLUSIONS: This study describes an outbreak of NDM-1-producing K. pneumoniae strains, isolated from a Tunisian hospital, caused by two clones belonging to ST147 and ST307; and highlights the role of OMPs loss, in combination with ß-lactamase expression, in conferring high carbapenem resistance.

Rectal Carriage of Extended-Spectrum Beta-Lactamase and Carbapenemase Producing Gram-Negative Bacilli in Intensive Care Units in Tunisia.

This study was conducted to evaluate the rate of fecal carriage of Gram-negative bacilli (GNB) resistant to third-generation cephalosporins (third GC) in patients hospitalized in the intensive care unit (ICU) of Charles Nicolle Hospital of Tunis and to identify the enzymatic mechanisms involved. From February to April 2014, rectal swabs were collected from all patients (n = 38) at admission and once weekly thereafter to identify acquisition. They were cultured on desoxycholate-lactose-agar plates supplemented with cefotaxime (2 mg/L). The rate of fecal carriage of GNB resistant to third GC was 0% (0/38) at admission and the acquisition rate was 45.16% (14/31). Nineteen GNB resistant to C3G were collected from 14 patients. The major species collected were Acinetobacter baumannii (n = 5), Klebsiella pneumoniae (n = 5), and Enterobacter cloacae (n = 5). Thirteen extended-spectrum ß-lactamase (ESBL) producing GNB were found; CTX-M-15 (n = 10) and CTX-M-14 (n = 1) among Enterobacteriacae and GES-12 (n = 2) among A. baumannii. Ten strains were carbapenem resistant. OXA-48 (n = 4) and NDM-1 (n = 1) were detected among Enterobacteriacae and OXA-23 (n = 5), and GES-11 (n = 1) were detected in A. baumannii. Gene encoding the ACT-16 AmpC-type-ß-lactamase was detected in two isolates. All Escherichia coli isolates were assigned to group B2. Among virulence genes, prevalence of fimH, fuyA, ompT, pai, and usp were highest observed in all E. coli isolates. Among K. pneumoniae mrkD and entB were the most frequent (n = 5) followed by ybtS (n = 4) and kfu (n = 2). This study revealed a high prevalence of fecal carriage of multidrug-resistant GNB, including ESBLs, carbapenemases, and cephalosporinases producing bacteria in patients hospitalized in ICU.

High Prevalence of Gut Microbiota Colonization with Broad-Spectrum Cephalosporin Resistant Enterobacteriaceae in a Tunisian Intensive Care Unit.

Healthcare-associated infections due to cefotaxime-resistant (CTX-R) Enterobacteriaceae have become a major public health threat, especially in intensive care units (ICUs). Often acquired nosocomially, CTX-R Enterobacteriaceae can be introduced initially by patients at admission. This study aimed to determine the prevalence and genetic characteristics of CTX-R Enterobacteriaceae-intestinal carriage in ICU patients, to evaluate the rate of acquisition of these organisms during hospitalization, and to explore some of the associated risk factors for both carriage and acquisition. Between December 2014 and February 2015, the 63 patients admitted in the ICU of Charles Nicolle hospital were screened for rectal CTX-R Enterobacteriaceae colonization at admission and once weekly thereafter to identify acquisition. CTX-R Enterobacteriaceae fecal carriage rate was 20.63% (13/63) at admission. Among the 50 non-carriers, 35 were resampled during their hospitalization and the acquisition rate was 42.85% (15/35). Overall, 35 CTX-R Enterobacteriaceae isolates were collected from 28 patients (25 Klebsiella pneumoniae, seven Escherichia coli, and three Enterobacter cloacae strains). Seven patients were simultaneously colonized with two CTX-R Enterobacteriaceae isolates. CTX-M-15 was detected in most of the CTX-R Enterobacteriaceae isolates (30/35, 88.23%). Three strains co-produced CMY-4 and 22 strains were carbapenem-resistant and co-produced a carbapenemase [OXA-48 (n = 13) or NDM-1 (n = 6)]. Molecular typing of K. pneumoniae strains, revealed eight Pulsed field gel electrophoresis (PFGE) patterns and four sequence types (ST) [ST101, ST147, ST429, and ST336]. However, E. coli isolates were genetically unrelated and belonged to A (n = 2), B1 (n = 2) and B2 (n = 3) phylogenetic groups and to ST131 (two strains), ST572 (two strains), ST615 (one strain) and ST617 (one strain). Five colonized patients were infected by CTX-R Enterobacteriaceae (four with the same strain identified from their rectal swab and one with a different strain). Whether imported or acquired during the stay in the ICU, colonization by CTX-R Enterobacteriaceae is a major risk factor for the occurrence of serious nosocomial infections. Their systematic screening in fecal carriage is mandatory to prevent the spread of these multidrug resistant bacteria.

High prevalence of extended-spectrum and plasmidic AmpC beta-lactamase-producing Escherichia coli from poultry in Tunisia.

This study was conducted to detect extended spectrum beta-lactamases (ESBLs) and plasmidic AmpC beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates in industrial poultry samples were collected from healthy chickens of the three farms. Samples were inoculated onto desoxycholate-lactose-agar plates supplemented with cefotaxime (2mg/L). E. coli was identified by biochemical and molecular methods and antibiotic susceptibility testing by the disk diffusion method. Genes encoding ESBLs and pAmpC-BL were detected by PCR and sequencing. Phylogenetic groups were determined by triplex PCR. The molecular typing of strains was done by pulsed field gel electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) in those isolates showing different PFGE patterns. Cefotaxime-resistant E. coli isolates were recovered in 48 of 137 fecal samples (35%), and one isolate/sample was further studied. The following beta-lactamase genes were detected: blaCTX-M-1 (29 isolates, isolated in all three farms), blaCTX-M-15 (5 isolates, confined in farm II), blaCTX-M-14 and blaCMY-2 (one isolate and 13 isolates, respectively, in farm III). The 48 cefotaxime-resistant isolates were distributed into phylogroups: B1 (n=21), A (n=15) and D (n=12). PFGE analysis revealed 19 unrelated patterns: 15 different profiles among ESBL-positive strains and 4 among the CMY-2-positive isolates. The following sequence types-associated phylogroups were detected: a) CTX-M-1-positive strains: lineages ST542-B1, ST212-B1, ST58-B1, ST155-B1 and ST349-D; b) CTX-M-15-positive strain: lineage ST405-D; c) CTX-M-14-positive strain: lineage ST1056-B1; d) CMY-2-positive strains: lineages ST117-D, ST2197-A, and ST155-B1. Healthy chickens constitute an important reservoir of ESBL- and pAmpC-BL-producing E. coli isolates that potentially could be transmitted to humans via the food chain or by direct contact.

Sequence variation in the E2-binding domain of HPV16 and biological function evaluation in Tunisian cervical cancers.

HPV16 E2 variants have different effects on the transcriptional activity of the LCR. In this study, we examined the nucleotide and amino acid sequence variation within the HPV16 E2 gene and to correlate with disease progression. E2 gene disruption was detected by PCR amplification of the entire E2 gene using a single set of primers. Nucleotide variations were analyzed by bidirectional sequencing. mRNA expression patterns of E6 and E7 gene transcripts were evaluated by a reverse transcriptase-PCR method (RT-PCR). The detection of intact E2 genes was significantly higher among controls than cases (81.8% versus 37.5%, resp., P < 0.05). Among the E subgroup, variation at position 3684 C>A results in the amino acid substitution T310K and was more common among the E2 undisrupted cases (7/9; 77.7%), compared to controls (2/9; 22.2%). In addition, specific sequence variations identified in the E2 ORF at positions 3684 C>A were associated with increased viral oncogenes E6-E7 production. Besides HPV16 E2 disruption, the 3684 C>A variation within undisrupted E2 genes could be involved in an alternative mechanism for deregulating the expression of the HPV16 E6 and E7 oncogenes and appears to be a major factor contributing to the development of cervical cancer in Tunisian women.

Characterization of extended spectrum ß-lactamase-producing Escherichia coli in community-acquired urinary tract infections in Tunisia.

This study was conducted to investigate the molecular epidemiology of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli in community-acquired (urinary tract) infections (CA-UTI) in Tunisia. Between January 2007 and December 2009, 15 E. coli isolates were collected at the laboratory of microbiology of Charles Nicolle Hospital of Tunis. Microbial identification was done with conventional methods. Antibiotic susceptibility was determined by disk diffusion method and ESBL detection was done with double-disk synergy test. ESBL typing was performed by polymerase chain reaction (PCR) and sequencing. Phylogenetic groups, virulence factors, and sequence type (ST)131 were determined by PCR. Genetic relatedness between strains was examined by pulsed-field gel electrophoresis (PFGE) after restriction with XbaI. The prevalence of ESBL-producing E. coli in CA-UTI was 0.046%. The majority of isolates were multidrug resistant. ESBL types were CTX-M-15 (n=13) and SHV-12 (n=2). The most common phylogenetic group was B2 (n=11) and virulence score was greater than or equal to 9 in nine strains. PFGE revealed 12 clusters. The majority of isolates (n=14) belonged to ST131 clone and 11 of them were CTX-M producers. In conclusion, this is the first detailed documentation of CA-ESBLs producing E. coli in Tunisia. Of particular concern is the emergence in our community of the highly diffusing CTX-M-15-B2-ST131 E. coli clone, which requires strengthening surveillance measures to countervail this emergent public health problem.

Characterization and molecular epidemiology of extended spectrum beta-lactamase producing Enterobacter cloacae isolated from a Tunisian hospital.

In 2009, out of the 66 nonrepetitive Enterobacter cloacae collected at Charles Nicolle hospital in Tunisia, 44 were extended spectrum ß-lactamase (ESBL) producers. The aim of the current study was to detect and characterize the genes encoding the ESBLs including blaTEM, blaSHV, and blaCTX-M groups by polymerase chain reaction and sequencing. Pulsed-field gel electrophoresis (PFGE) analysis was used to determine the genetic relatedness between isolates. All strains were susceptible to carbapenems. They were resistant to fluoroquinolones, gentamicin, tobramycin, and trimethoprim+sulfamethoxazole but variably resistant to netilmicin, amikacin, and tetracyclines. Sequence analysis of the polymerase chain reaction products revealed the presence of blaCTX-M-15 (39 strains), blaSHV-12 (6 strains), and blaSHV-27 (1 strain). The coexistence of two ESBLs was observed in two isolates harboring, respectively, SHV-12+CTX-M-15 and SHV-27+CTX-M-15. PFGE revealed 36 unrelated profiles. Diffusion of E. cloacae producing CTX-M-15 ESBL in our hospital is the consequence of dissemination of identical or related plasmids harboring the CTX-M-15 gene.

Carbapenem-resistant Acinetobacter baumannii producing the carbapenemase OXA-23 in Tunisia.

AIM: To analyze the mechanisms of resistance to carbapenems among imipenem resistant A. baumannii recovered from different wards at Charles Nicolle Hospital. METHODS: From January to December 2007, 50 carbapenem-resistant A. baumannii isolates were recovered from hospitalized patients. MICs were performed by agar dilution method and interpreted according to CLSI guidelines. Metallo-ß-lactamase production was evaluated using imipenem-EDTA disk synergy test. PCR and DNA sequencing targeting blaOXA genes were performed and pulsed field gel electrophoresis was used for epidemiologic study. RESULTS: Most of the isolates were obtained from patients hospitalized in surgery (62%) and Intensive Care Units (22%). All strains showed high level of resistance to ticarcillin (MIC50 > 2048µg/ml), ticarcillin-clavulanic acid (MIC50 >1024µg/ml), aztreonam (MIC50 = 512µg/ml), ceftazidim (MIC50 = 512µg/ml), imipenem (MIC50 = 512µg/ml), meropenem (MIC50 =128µg/ml) and cefepime (MIC50 = 256µg/ml). Metallo-ß-lactamase production was negative for all isolates. The co-existence of blaOXA-51-like/ blaOXA-23-like was detected in 82% (n= 41). The genes blaOXA- 24-like and blaOXA-58-like were not found in any isolate. All isolates harboured a blaOXA-51-like gene. Sequencing confirmed the presence of blaOXA-23 and blaOXA-69 genes. Eight distinct patterns were observed (A: 41 isolates, B: 1 isolate, C: 1 isolate, D: 1 isolate, E: 1 isolate, F: 2 isolates, G: 1 isolate, H: 2 isolates). CONCLUSION: Production of OXA-23 was the important mechanism of resistance to carbapenem among A. baumannii. Strengthening of prevention measures are required to control further spread of carbapenemases in Tunisia.

Hospital acquired outbreak of methicillin-resistant Staphylococcus aureus infection initiated by a health care worker.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become an increasingly important pathogen leading to hospital acquired infections. AIM: This study was done to confirm an outbreak of MRSA suspected at Charles Nicolle Hospital. METHODS: From 26 April to 11 June 2002, six patients hospitalized in the dermatologic ward at Charles Nicolle hospital of Tunisia have developed infections caused by MRSA. An investigation of the outbreak has been detected a nasal carriage nurse. This carrier received topical mupirocin treatment to decolonize the anterior nares and the outbreak was stopped without further incident. RESULTS: Typing of the MRSA strains by pulsed field gel electrophoresis demonstrated the same pulsotype shared by all isolates showing that MRSA isolates belonged to a single clonal type responsible of outbreak. Colonized nurse was the source of MRSA dissemination. CONCLUSION: This report illustrates the risk of nosocomial outbreak linked to cares delivered by the staff personnel. More sensibilisation and the respect of strict hygienic measures should be emphasized.

Clonal spread of carbapenem resistant Pseudomonas aeruginosa in an university hospital.

AIM: We examined 14 Pseudomonas aeruginosa clinical isolates collected in 2000 from patients hospitalised in different wards at Charle Nicolle hospital from Tunisia. METHODS: Analysis includes serotyping, antimicrobial susceptibility profile, beta-lactamase detection, randomly amplified polymorphic DNA and pulsed field gel electrophoresis. All Pseudomonas aeruginosa strains belonged to serotype O12 and they demonstrated a high level of resistance to all antibiotioc tested. RESULTS: Beta-lactamase detection revealed that 9 of these strains had ceftazidime activity restored by cloxacillin and none of the 14 strains were metallo-beta-lactamase producing. Randomly amplified polymorphic DNA analysis and pulsed field gel electrophoresis were able to discriminate isolates and gave concordant results which showed epidemiologically related strains. CONCLUSION: These results confirm a clonal spread of multiresistant Pseudomonas aeruginosa O12 throughout the hospital.

Spread of multidrug resistant Acinetobacter baumannii in a teaching hospital.

BACKGROUND: A. baumannii is an important opportunistic pathogen widely distributed in the hospital environment and responsible for a variety of nosocomial infections especially in patients from intensive care units. AIM: We describe an outbreak of Acinetobacter baumannii (16 stains) in 3 intensive care units (I, II, III) at Charles Nicolle hospital of Tunis over a 5 month period (March to July 2005). METHODS: The antimicrobial susceptibility was determined by disc diffusion test and the genetic relatedness of isolates was done by Random Amplified Polymorphic DNA (RAPD) analysis. Two strains not related to the outbreak were used for the discrimination of the technique. RESULTS: Samples were collected from blood (44%), materials (31%), pus (6.5%), urines (6.5%) and respiratory tract (12.5%). Antibiotic resistance pattern showed 2 different profiles. However, molecular typing of isolates revealed 3 distinct profiles (A, B, C) represented respectively by 8, 7 and one isolates. The major profile was the profile A found in 5 patients and in materials. It was appeared firstly in intensive care unit I, then in the 2 other units (II and III). The profile B was observed also in the 3 units. However, the profile C was found in one patient in unit I. CONCLUSION: These data emphasize the need for active surveillance for multidrug-resistant Acinetobacter baumannii, and the value of molecular typing of strains in hospital settings to investigate spread of infection.