Gut acquisition of Extended-spectrum ß-lactamases-producing Klebsiella pneumoniae in preterm neonates: Critical role of enteral feeding, and endotracheal tubes in the neonatal intensive care unit (NICU).

BACKGROUND: Klebsiella spp. can colonize the intestine of preterm neonates, and over-growth has been associated with necrotizing enterocolitis, hospital-acquired infections, and late-onset sepsis. This could lead us to suggest that the clinical pertinence of intestinal colonization with ESBL in preterm neonates appears to be important. We conducted this study to characterize the genetic proprieties of ESBL-producing Klebsiella pneumoniae (ESBL-KP) under clinical isolates and to describe the risk factors for the intestinal tract acquisition event during hospitalization. METHODS: One hundred and thirteen premature infants were recruited from the neonatal intensive care unit (NICU). All newborns are issued from the birth suites of the pregnancy department. Two rectal swabs were planned to define K. Pneumoniae intestinal carriage status. ESBL-KP was confirmed by Brilliance ESBL selective chromogenic Agar. Antimicrobial susceptibility testing including phenotypic testing and genotypic detection of the most commonly described ESBL genes was done. Logistic regression models were performed to find the variables associated with the acquisition event of ESBL-KP. RESULTS: A total of 62 (54.86%) premature neonates were colonized with ESBL-KP. The rate of blaSHV, blaTEM, blaCTX-M1, blaCTX-M2, blaCTX-M9, and blaOXA-48 genes among the isolates was 82, 48, 93.5, 4.8, 11.2 and 3.22%, respectively. We found that ESBLs K. Pneumoniae isolates were 100% resistant to amoxicillin, clavulanic acid-amoxicillin, cefotaxime, ceftazidime, and gentamicin. The regression model is for a given significant association between the tract intestinal of ESBL-KP acquisition events and the use of enteral tube feeding (OR = 38.46, 95% CI: 7.86-188.20, p-Value: 0.001), and endotracheal tubes (OR = 4.86, 95% CI: 1.37-17.19, p-Value 0.014). CONCLUSION: Our finding supposes that the enteral feeding tube and endotracheal tube might have a critical role in colonizing the intestinal tract of preterm infants. This highlights the current status of both practices that will require updated procedures in the NICU.

Acinetobacter baumannii Carbapenemase Producers in Morocco: Genetic Diversity.

INTRODUCTION:  Carbapenem resistance in Acinetobacter baumannii (A. baumannii) is a public health problem worldwide. Although carbapenem resistance is emerging in Morocco, few studies have shown the epidemiological profile of carbapenemase genes in Moroccan healthcare facilities. The aim of this study was to characterize the molecular profile of the carbapenemase enzyme in Acinetobacter baumannii from clinical isolates. METHODS: Clinical strains isolated in the laboratory from various samples were subjected to several phenotypic tests. Antibiotic susceptibility and identification were tested using Phoenix 100 (Becton Dickinson Co., Sparks, MD, USA) and Api 20 (bioMérieux, Marcy-l'Etoile, France). Simple phenotypic assays were used to detect carbapenemase oxacillinase (OXA) and metallo-ß-lactamase (MBL) production, including the modified Hodge test (MHT) and ethylenediaminetetraacetic acid (EDTA) test. The detection of carbapenemase genes was performed by multiplex and simple polymerase chain reaction (PCR). RESULTS: A total of 140 strains or 100% of isolates contained OXA-51 and ISbA1 sequences, 89% contained OXA-23 and OXA-58 sequences, and 1% contained OXA-24 sequence. The MBL genes were predominated by Verona integron-encoded metallo-ß-lactamase (VIM) (56%), followed by Seoul imipenemase (SIM) (39%), German imipenemase (GIM) (37%), São Paulo metallo-ß-lactamase (SPM) (13%), imipenemase (IMP) (11%), and New Delhi metallo-ß-lactamase (NDM) (4%). Guyana extended-spectrum ß-lactamase (GES) was not found in any isolation. CONCLUSION: Our study shows a high frequency of carbapenem resistance in Acinetobacter baumannii, as it reports a high molecular diversity of carbapenemase-encoding genes, mainly dominated by the carbapenemase ISaba1/OXA-23, which represents an emerging threat in our hospital.

Intense Intestinal Carriage of Carbapenemase-Producing Klebsiella pneumoniae Co-harboring OXA-48, KPC, VIM, and NDM Among Preterm Neonates in a Moroccan Neonatal Intensive Care Unit.

INTRODUCTION: This study aimed to investigate the prevalence and the carbapenemase production ability of Klebsiella pneumoniae isolates from premature neonates' intestinal tracts in a Moroccan neonatal intensive care unit Methodology: Active rectal screening was performed among 339 preterm infants. The collected isolates were subjected to antibiotic susceptibility testing, phenotypic analysis of carbapenemase production, and molecular detection of carbapenemase genes. RESULTS: Out of 293 K. pneumoniae isolates collected, 31.05% (91) were resistant to carbapenem and produced carbapenemase, resulting in a 22.12% rate of intestinal carriage. Among the carbapenem-resistant K. pneumoniae isolates, 40.65% (37) had co-harbored carbapenemase genes. All isolates contained the blaOXA-48 gene, and the blaNDM, blaVIM, and blaKPC genes were detected in 30.76%, 9.89%, and 2.19% of the isolates, respectively. Out of 30.76% of these isolates had both the blaOXA-48 and blaNDM genes, 8.79% had both blaOXA-48 and blaVIM, and only 2.20% had both blaOXA-48 and blaKPC genes. Furthermore, 88.57% of carbapenem-resistantK. pneumoniae isolates co-harboring carbapenemase genes were genetically related strains. CONCLUSIONS: This study revealed a high prevalence of intestinal carriage of carbapenem-resistant K. pneumoniae. Therefore, implementing effective screening and diagnostic measures, and focusing on antimicrobial stewardship are essential to preventing the spread of these resistant strains and minimizing the risk they pose to premature infants.

Paediatric osteoarticular infections caused by staphylococcus aureus producing panton-valentine leucocidin in morocco: Risk factors and clinical features.

OBJECTIVE: We aimed to estimate the prevalence of Staphylococcus aureus producing Panton-Valentine leucocidin (PVL) isolated from children diagnosed with osteoarticular infections (OAIs), and to examine risk factors and clinical features. METHODS: This prospective study was conducted from January 2017 to December 2018. All hospitalised children diagnosed with S. aureus OAI are included. Blood cultures, articular fluids, synovial tissues and/or bone fragments were collected for bacteriological culture. Antimicrobial susceptibility tests were determined by disk diffusion method. Genes encoding methicillin resistance (mecA) and PVL virulence factors (luk-S-PV and luk-F-PV) were detected by multiplex polymerase chain reaction. The demographic, clinical, laboratory, radiographic and clinical features were reviewed prospectively from medical records. RESULTS: A total of 37 children with S. aureus OAIs were included, 46% of them have PVL-positive infection and 70.6% were male. The mean age was 8.12 years (±4.57), and almost were from rural settings (76.5%). Children with Staphylococcus aureus producing Panton-Valentine leucocidin (SA-PVL) were significantly associated with type of infection (P = 0.005), location of infection (P = 0.037) and abnormal X-ray (P = 0.029). All strains SA-PVL+ are sensitive to methicillin, but one strain SA-PVL negative was methicillin-resistant S. aureus, confirmed by gene mecA positive. CONCLUSION: The prevalence of S. aureus infections producing PVL toxin was high in OAIs amongst Moroccan children, mainly due to methicillin-susceptible S. aureus. Type and location of infections and abnormal X-ray were significantly associated with SA-PVL. Routine diagnostic testing of PVL-SA, continuous epidemiological surveillance and multidisciplinary management of OAI is essential to prevent serious complications.

First report of SPM metallo-ß-lactamases producing Acinetobacter baumannii isolates in Morocco.

Background and Objectives: Carbapenem-resistant Acinetobacter baumannii has recently been identified by the World Health Organization as a critical pathogen. We propose to characterize the molecular characteristics of clinical isolates of A. baumannii resistant to carbapenems collected in a Moroccan hospital. Materials and Methods: Seventy carbapenem-resistant A. baumannii isolates from various samples were received at the microbiology laboratory of the Hospital Center. Antibiotic susceptibility was tested by the diffusion disc method and molecular characterization of antimicrobial resistance was performed by PCR and sequencing. Results: Carbapenemase genes were detected in our isolates: the OXA-51 gene and the ISbA1 sequence were detected in all isolates (100%), the OXA-23 and OXA-58 genes were detected in 82.85% and 10% of isolates respectively, MBL genes were dominated by VIM 39 isolates (55.7%), followed by GIM 26 isolates (37%), SIM 20 isolates (28.5%), IMP 8 isolates (11, 4%), NDM 3 isolates (4%) and for the first time in Morocco SPM with 4 isolates (5.7%). Conclusion: The emergence of resistance of A. baumannii to carbapenems is a serious problem in our hospital which requires the establishment of a prevention strategy and strict respect for hygiene to minimize their dissemination.

First report of Kingella kingae diagnosed in pediatric bone and joint infections in Morocco.

BACKGROUND: The progress of diagnostic strategies and molecular methods improved the detection of Kingella kingae in bone and joint infections, and now, Kingella kingae is being increasingly recognized as the most frequent cause of bone and joint infection BJI in early childhood. The main objective of this prospective study is to report the frequency of Kingella Kingae in negative culture bone and joint pediatric infections, and to describe the clinical and biologic features of these children. METHODS: From December 2016 to June 2019, we selected all hospitalized patients with suspected BJI. When culture was negative on the fifth day, children under 10 years were subsequently included in the study, and PCR assay was performed systematically for researching K. kingae specific gene cpn60. Microbial culture and identification were made using standard bacteriological methods. The demographics, clinical, laboratory, radiographic and clinical features were reviewed from medical records. RESULTS: We enrolled 65 children with culture negative BJI, 46 of them having under 10 years old have been screened for the cpn60 gene. Thus, the gene encoding Kingella kingae was positive for 27 BJI cases (58.7%). The mean age of children was 3.02 years, 55.6% were aged 6 months-4 years and 29.6% of them were aged 5-10 years. The male to female ratio was 1.7 and 16 cases (59.26%) occurred during the fall-winter period. The most frequent BJI type was septic arthritis (77.8%) and the most affected sites were knee (51.9%) and hip (37.0%). We recorded a mild clinical picture with normal to mildly raised inflammatory markers. All patients had good clinical and functional outcomes, with no serious orthopedic sequelae.. CONCLUSION: K kingae is an important pathogen of culture-negative BJI in Moroccan children. PCR testing should be performed in culture-negative cases of children not only in the typical age range of 6 months to 4 years. When implemented in the routine clinical microbiology laboratory, a specific K. kingae PCR assay can provide a better diagnostic performance of BJI.

Intense intestinal carriage and subsequent acquisition of multidrug-resistant enterobacteria in neonatal intensive care unit in Morocco.

This study was conducted in order to know the colonization rate of MDR enterobacteria in neonates during their hospitalization in neonatal intensive care unit (NICU). Furthermore, we investigated risk factors for potential colonization and molecular epidemiology of isolated resistant bacteria. This prospective study was carried out in the neonatology and intensive care unit department of the University Hospital of Fez (Morocco) from February 2013 to July 2015. All consecutive admitted newborns were screened for intestinal and nasal carriage of MDR enterobacteria at admission of the babies and during the hospitalization. During the study period, a total of 641 Enterobacteriaceae were isolated and Klebsiella pneumoniae was the predominated bacteria. Bacterial identification and antibiograms were performed according to the international standards. On admission, 455 newborns were screened. A median age of these newborns was 1 day with an extended 147 days and their average weight was 2612 ± 1023 grams. 22.4% of neonates were found colonized by an ESBL producing Enterobacteriaceae (ESBL-E), 8.7% by a carbapenemases producing Enterobacteriaceae (CPE). During hospitalization, 207 of newborns were included in the acquisition study. 59.4% of newborns acquired an ESBL-E during their stay, 12.5% has acquired CPE. The blaCTXM-15 gene was the most frequently detected (81.2%) among ESBL-E. While, all CPE has expressed the blaOXA-48 gene exclusively. Two risk factors have been significantly associated with MDR enterobacteria colonization at admission which are newborns admission from maternity of the university hospital (95% CI, 1.859-5.129, P = 0.000) and neurological distress (95% CI, 1.038 to 4.694, P = 0.040). During hospitalization, the none risk factor was significantly associated with the carriage of MDR-E. The high rate of colonization, the MDR enterobacteria and the resistance genes found represent good indicator of cross-transmission in the NICU. An active strategy to control the spread of MDR enterobacteria should be applied.

Detection of metallo-beta lactamases and oxacillinase genes in carbapenem-resistant Acinetobacter baumannii strains isolated in Morocco.

INTRODUCTION: resistance to carbapenem is widespread among Acinetobacter baumannii (A. baumannii) strains. Metallo-beta lactamases enzymes (MBL) are responsible for carbapenem resistance, as are oxacillinases (OXA). In recent years, MBL producing carbapenem-resistant strains have been reported in the world and Morocco at increasing rates. Our study aimed to investigate the presence of carbapenemases in acinetobacter strains isolated from hospitalized patients in CHU Fez. METHODS: a total of 58 imipenem-resistant A. baumannii strains isolated from clinical samples were investigated. The presence of MBL was described phenotypically by the double-disk synergy test (DDST), MBL E-test, and modified Hodge test. The blaIMP, blaVIM, genes, and blaOXA-23, blaOXA-51 genes were investigated by multiplex polymerase chain reaction (PCR). The blaNDM-1 gene was determined by simplex PCR. RESULTS: fifty-eight strains were resistant to imipenem (98%), the modified Hodge test (MHT) was positive for 58 strains (100%), 47 strains (82%) were found to be positive for MBL by the test of double-disk synergy (DDST), 58 strains (100%) were positive by E-test MBL. The OXA 51 gene was detected in all strains, and the OXA 23 gene was detected in 53 strains (91%). In addition, the MBL genes were not detected by genotypic methods. CONCLUSION: the OXA-23 and OXA-51 carbapenemases type are responsible for the resistance to carbapenems in A. baumannii resistant to carbapenems in our establishment. Resistance to carbapenems by MBL enzymes has been found by phenotypic tests, which must be confirmed by genotypic methods; and solicit other MBL genes.

Evaluation of Antibacterial Activity of Three Quaternary Ammonium Disinfectants on Different Germs Isolated from the Hospital Environment.

BACKGROUND: The microbiological risk of the hospital environment, including inert surfaces, medical devices, and equipment, represents a real problem. OBJECTIVE: This study is aimed at demonstrating and assessing the antibacterial activity of three synthetic disinfectants classified as quaternary ammoniums on different bacterial strains (Gram-negative and Gram-positive like Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus) isolated from the hospital environment. The reference strains included Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853 used as negative control strains. METHOD: Three quaternary ammonium disinfectants were tested: DDN9® (0.5%) which contains didecylmethylpolyoxyethylammonium propionate as an active substance, spray (0.4%) containing quaternary ammonium compounds, and Phagosurf ND® (0.4%) with didecyldimethylammonium chloride. Their effect was evaluated using the disk diffusion technique and the broth dilution methods, allowing the Minimum Inhibitory Concentration (MIC) and then the Minimum Bactericidal Concentration (MBC). RESULT: Only the growth of Gram-positive bacteria and some strains of Gram-negative bacteria were inhibited by the three synthetic disinfectants. NDD9® demonstrated an antibacterial effect only against the Gram-positive strains (S. aureus and S. aureus ATCC 29213) with a MIC of 0.25 mg/ml. The disinfectant spray showed effect against all four strains including E. coli (9), S. aureus, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 with an inhibitory concentration of 4 mg/ml, while the growth of S. aureus ATCC 29213 was inhibited at 2 mg/ml. The third disinfectant, Phagosurf ND®, inhibited only the growth of S. aureus ATCC 29213 at a MIC of 4 mg/ml. CONCLUSION: This study is the first here in Morocco to evaluate the bacterial activity of products intended for the control of the healthcare environment. The results obtained on the three disinfectants tested reveal an ineffectiveness against some isolated strains from the hospital environment.

The Antistaphylococcal Activity of Amoxicillin/Clavulanic Acid, Gentamicin, and 1,8-Cineole Alone or in Combination and Their Efficacy through a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Osteomyelitis.

The aim of this research paper is to test the antistaphylococcal effect of 1,8-cineole, amoxicillin/clavulanic acid (AMC), and gentamicin, either separately or in combination against three Staphylococcus aureus strains isolated from patients suffering from osteomyelitis. This activity was tested in vitro by using the microdilution method and the checkerboard assay. The efficacy of these three antibacterial agents was then tested in vivo by using an experimental model of methicillin-resistant S. aureus osteomyelitis in rabbits. This efficacy was assessed after four days of treatment by counting the number of bacteria in the bone marrow. The obtained results in vitro showed that the combination of the AMC with gentamicin did not induce a synergistic effect, whereas the combination of the two antibiotics with 1,8-cineole did. This effect is stronger when AMC is combined with 1,8-cineole as a total synergistic effect was obtained on the three strains used (FIC ≤ 0.5). In vivo, a significant reduction was noted in the number of colonies in the bone marrow when rabbits were treated with AMC associated with either 1,8-cineole or gentamicin compared to rabbits treated with AMC, gentamicin, or 1,8-cineole alone. These results demonstrated that 1,8-cineole showed a synergistic effect in combination with both AMC and gentamicin, which offer possibilities for reducing antibiotic usage. Also, the AMC associated with 1,8-cineole could be used to treat MRSA osteomyelitis.

Intestinal carriage of antibiotic resistant Acinetobacter baumannii among newborns hospitalized in Moroccan neonatal intensive care unit.

This study was conducted in order to assess the acquisition rate of Acinetobacter baumannii by newborn screening, on admission and during the discharge process of neonatal intensive care unit. (NICU). Furthermore, we investigated risk factors for potential colonization and molecular epidemiology of isolated resistant bacteria. This prospective study was conducted in the neonatal unit of Hassan II University Hospital of Fez from February 2013 to July 2015. During this period, all consecutive admitted neonates were screened for A. baumannii intestinal carriage, on admission and during the discharge process. Bacteriological and molecular tests were evaluated according to the international standards. This study examines the screening on admission of 455 newborns, 59% of whom were male. The average gestational age and birth weight were 35.2 weeks and 2612.1 g respectively. In total, 277 patients were included in the acquisition study on admission. The prevalence of multi-drug resistant (MDR) A. baumannii strain carriage was 6.5%, while the acquisition rate during the hospital recovery was 13.7%. In this study, 68 MDR A. baumannii isolates were collected. The resistance rates to different antibiotic classes including, Ceftazidime, Gentamycin and Ciprofloxacin varied between 92 and 100%. Moreover, 13% of MDR A. baumannii isolates were carbapenemase producers and 88% harbored blaOXA-23 gene. On admission, three risk factors were significantly associated with A. baumannii colonization: age (OR, 2.803; IC95%, 1.191-6.596; P = 0.01), gender (OR, 0.382; IC95%, 0.158-0.921; P = 0.03) and the delivery birth at the Maternity of University Hospital (MUH), (OR, 0.196; IC95%, 0.071-0.540; P = 0.002). However during hospitalization, the only risk factor associated with acquisition of A. baumannii was the respiratory distress (OR, 2.270; IC95%, 1.055-4.881; P = 0.03). A high intestinal carriage rate of A. baumannii and multiple antibiotic resistance were found in our NICU. Thus, the spread of MDR A. baumannii should be monitored by an active surveillance strategy.

Rectal carriage of extended-spectrum ß-lactamase- and carbapenemase-producing Enterobacteriaceae among hospitalised neonates in a neonatal intensive care unit in Fez, Morocco.

OBJECTIVES: The aim of this study was to investigate the faecal carriage and molecular epidemiology of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBLE) isolated from rectal samples of neonates hospitalised in a neonatal intensive care unit (NICU) of a university hospital in Fez, Morocco. METHODS: From February-July 2013, all neonates hospitalised in the NICU were screened for ESBLE carriage at discharge. ESBLs were identified by double-disk synergy test, PCR and DNA sequencing analysis. ESBLE were analysed by pulsed-field gel electrophoresis (PFGE), and conjugation was performed by the broth mating method. RESULTS: In this study, 169 Enterobacteriaceae were collected from 164 neonates. The prevalence of faecal carriage of ESBLE was 58.0% (98/169), predominantly Klebsiella pneumoniae (65/98; 66.3%). A high rate of multiresistance in ESBLE was noted. blaCTX-M-1 group (78.5%) was the most frequent ESBL gene detected, and all isolates harboured the CTX-M-15 variant. The prevalence of carbapenemase-producing Enterobacteriaceae was 1.8%, and blaOXA-48 was the only gene found in these isolates. Sequencing revealed subgroups corresponding to bla(CTX-M-15,TEM-1,TEM-104,SHV-1,SHV-44,SHV-49andSHV-133) genes. Conjugation experiments showed the transferability of blaCTX-M-15 and blaTEM, but not blaSHV. These genes were carried by a high-molecular-weight conjugative plasmid (ca. 125kb). PFGE profiles demonstrated high clonal dissemination of ESBL-positive strains in the NICU. CONCLUSIONS: These results demonstrate the existence of high clonal transmission of ESBLE in a Moroccan NICU. This finding provides useful information to implement a screening policy for resistant Enterobacteriaceae among neonates hospitalised in this ward.

Prevent infection linked to the dialysis water in a hemodialysis center in Fez city (Morocco).

BACKGROUND: Water treatment systems are a critical variable in dialysis therapy. Rigorous control of hemodialysis water quality is particularly important in order to guarantee a better quality of life of the hemodialysis patients. The objective of the study was to evaluate the chemical, microbiological quality and antimicrobial resistance of bacteria isolated from water and dialysate in a public HD center. METHODS: Fifty five samples of water and dialysate were collected weekly over a period of 4 months. The samples were collected from 4 points in the distribution loop. The microbiological and chemical analyses were performed according to our national standards. Antimicrobial susceptibilities patterns of isolated bacteria were determined by disk diffusion method. RESULTS: The chemical and microbiological parameters in all dialysis water and dialysate samples are in accordance with national standards. However, 70 Gram-negative bacteria were identified: Pseudomonas sp, Ochrobactrum antropi and Burkholderia cepacia, isolated at 52.8%, 12.8% and 17% simultaneously. Fourteen per cent of the isolates were resistant to three or more antibiotics. All resistant bacteria belong to the genus of Pseudomonas, 80% were resistant to tetracycline and to co-trimoxazole, 30% to ceftazidime. No colistin and imipenem resistance was observed. CONCLUSION: To avoid a health risk due to bacterial contamination, an adequate system for water treatment, disinfection of the hemodialysis system and microbiological monitoring of the water and dialysate are necessary.

Staphylococcus aureus nasal carriage in a Moroccan dialysis center and isolates characterization.

Staphylococcus aureus, which has its ecological niche in the anterior nares, has been shown to cause a variety of infectious diseases mainly for patients in hemodialysis units. We performed this study to evaluate the prevalence of nasal S. aureus carriage among hemodialysis outpatients, to determine the antimicrobial susceptibility of isolates, to characterize the virulence genes, and to identify associated risk factors. Nares swab specimens were obtained from 70 outpatients on hemodialysis between March and June 2010. Samples were plated immediately onto S. aureus specific media and pattern of antibacterial sensitivity was determined using disk diffusion method. Polymerase chain reaction was used to detect nuc, mecA, and genes encoding staphylococcal toxins. Medical record of patients was explored to determine S.aureus carriage risk factors. Nasal screening identified 42.9% S. aureus carriers with only one (3.3%) methicillin-resistant S. aureus isolate. Among the methicillin-susceptible S. aureus isolates, high rate of penicillin resistance (81.8%) has been detected. The identified risk factors were male gender and age ≤ 30 years. Research of virulence factors showed a high genetic diversity among the 30 S. aureus isolates. Twenty-one (70%) of them had at least one virulence gene, of which 3.3% were Panton-Valentine leukocidin (lukS/F-PV) genes. S. aureus carriage must be screened for at regular intervals in hemodialysis patients. Setting up a bacterial surveillance system is one of the strategies to understand the epidemiology of methicillin-resistant S. aureus, to guide local antibiotic policy and prevent spread of antibiotic-resistant S. aureus.