Wafaa Jamal

Emergence of CTX-M-15 type extended-spectrum β-lactamase-producing Salmonella spp. in Kuwait and the United Arab Emirates

Cephalosporins are major antimicrobials used to treat serious Salmonella infections. However, their effectiveness is being compromised by the emergence of extended-spectrum beta-lactamases (ESBLs). The genetic determinants encoding ESBL in Salmonella spp. isolated from patients in Kuwait and United Arab Emirates (UAE) were studied over a 2 year period. Out of a total of 407 isolates, 116 isolates possessed the resistance phenotypes consistent with possible ESBL production. Of these, 69 (59.5 %) were ESBL positive. PCR and sequencing were used to determine the genetic determinant(s) responsible for ESBL phenotypes. A total of 14 (12.1 %) and 29 (24.6 %) isolates were CTX-M-15 ESBL producers and TEM producers, respectively. Ten CTX-M-15 producers carried the insertion sequence ISEcpI gene. PFGE analysis revealed identical profiles in 4 of the 13 Kuwaiti strains. This study reports the presence of the bla(CTX-M-15) gene in Salmonella spp. and Salmonella enterica serotype Typhi from Kuwait and UAE for what is believed to be the first time. This is of great concern as the gene is also found in association with the ISEcpI gene, which may easily facilitate its spread. These isolates originated mostly from non-Kuwaiti Arabs rather than from people of Asian origin.

Spectrum and antibiotic resistance of uropathogens isolated from hospital and community patients with urinary tract infections in two large hospitals in Kuwait.

Objectives: To determine the spectrum of microbial etiology and antibiotic resistance pattern of the uropathogens that cause urinary tract infections in 2 large teaching hospitals in Kuwait over a period of 1 year. Materials and Methods: The Vitek identification card system was used to identify the uropathogens. Susceptibility of the isolates against 18 antibiotics was performed by the microbroth dilution method using the Vitek automated system. In addition, gram-positive bacteria were tested in parallel by the disk diffusion technique. Results: The six overall most common isolates were: Escherichia coli, accounting for 47% of isolates in both hospitals, followed by Candida spp. (10.8%), Klebsiella pneumoniae (9.6%), Streptococcus agalactiae (GBS; 9.5%), Enterococcusfaecalis (4.2%) and Pseudomonas aeruginosa (4.1%). Amikacin provided the widest coverage amongst all the antibiotics tested followed by ciprofloxacin, gentamicin and piperacillin-tazobactam. For the gram-negatives, high resistance (26–63%) to the β-lactam antibiotics was noted, especially to ampicillin, amoxicillin-clavulanic acid, cephalothin and cefuroxime. Resistance to trimethoprim-sulfamethoxazole was also high. None of the enterococci was resistant to the glycopeptides, but 38–60% of the Staphylococcus haemolyticus were resistant to vancomycin or teicoplanin. Conclusion: These data show the high level of antimicrobial resistance amongst the uropathogens causing urinary tract infection in the two hospitals studied.

Comparison of two matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry methods and API 20AN for identification of clinically relevant anaerobic bacteria

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid microbial diagnosis at relatively low cost and can be considered an alternative to conventional biochemical and molecular identification systems in clinical microbiological laboratories, including anaerobe laboratories. Two commercially available MALDI-TOF MS systems, Bruker Microflex MS and bioMérieux VITEK MS, were evaluated for the identification of 274 isolates of clinically significant anaerobic bacteria recovered from routine cultures of clinical specimens in parallel with blinded comparison with conventional biochemical (API 20AN) or molecular methods. All were recovered cultures obtained from patients attending the Mubarak Al Kabir Hospital, Kuwait, during a 6 month period. Discrepant results after two attempts at direct colony testing had failed to provide acceptable MALDI-TOF identification were resolved by gold-standard 16S gene sequencing. The VITEK MS gave high-confidence identification of the 274 isolates, all of which were correctly identified. The Bruker Microflex MS system also gave high-confidence identification for 272 of the 274. After discrepancy testing, the Bruker MS results agreed with biochemical or molecular methods for 89.1 % of the isolates at species level and 10.2 % at genus level (0.72 % were misidentified). The level of agreement with the VITEK MS was 100 % at both species and genus level; no isolates were misidentified. Our data suggest that implementation of MALDI-TOF MS as a first step for identification will shorten the turnaround time and reduce the cost in the anaerobe laboratory.

Rapid identification of pathogens directly from blood culture bottles by Bruker matrix-assisted laser desorption laser ionization-time of flight mass spectrometry versus routine methods

The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of microorganisms directly from blood culture is an exciting dimension to the microbiologists. We evaluated the performance of Bruker SepsiTyper kit™ (STK) for direct identification of bacteria from positive blood culture. This was done in parallel with conventional methods. Nonrepetitive positive blood cultures from 160 consecutive patients were prospectively evaluated by both methods. Of 160 positive blood cultures, the STK identified 114 (75.6%) isolates and routine conventional method 150 (93%). Thirty-six isolates were misidentified or not identified by the kit. Of these, 5 had score of >2.000 and 31 had an unreliable low score of <1.7. Four of 8 yeasts were identified correctly. The average turnaround time using the STK was 35 min, including extraction steps and 30:12 to 36:12 h with routine method. The STK holds promise for timely management of bacteremic patients.

In vitro activity of 15 antimicrobial agents against clinical isolates of Clostridium difficile in Kuwait

A total of 73 clinical isolates of Clostridium difficile isolated from stool/rectal swabs of patients admitted to the intensive care units at Mubarak Hospital, Ibn Sina Hospital Burn unit and Haematology wards at the Kuwait Cancer Control Centre, were investigated for their susceptibility to 15 antibiotics using the Etest. Amoxycillin-clavulanic acid, ampicillin, meropenem, metronidazole, penicillin, piperacillin, piperacillin/tazobactam, teicoplanin and vancomycin had excellent activities with MIC(90)s of 0.38, 0.5, 1, 0.19, 1.5, 2, 3, 0.25 and 0.75 mg/l, respectively. Of the 73 C. difficile isolates, 86% were resistant to imipenem (MIC(90) >32 mg/l) and almost 97% were resistant to trovafloxacin (MIC(90)>256 mg/l). Forty eight percent of the isolates were resistant to clindamycin. A total of 18 isolates were highly clindamycin-resistant with an MIC of >256 mg/l; 10 of these were toxin producers. Multiple antibiotic resistance (two or more antibiotics) was noted in 63 isolates. These were more common among the toxigenic strains than the non-toxigenic strains by a ratio of 2.5:1.

Role of tigecycline in the control of a carbapenem-resistant Acinetobacter baumannii outbreak in an intensive care unit.

The incidence of Acinetobacter baumannii infection has greatly increased over recent decades with infections occurring more in critically ill hospitalised patients. Hospital outbreaks of multiple antibiotic-resistant strains are posing an increasing threat to public health. Three different outbreaks of multidrug-resistant A. baumannii (MRAB) infections involving 24 patients, aged 16-75 years occurred in the intensive care unit in the course of one year. The isolates were cultured from clinical samples and identified using automated Vitek II ID system and the API 20NE system. Susceptibility testing was done by the E-test method. Molecular typing of the isolates was determined by pulsed-field electrophoresis. Screening of both patients and the environment was carried out. The acquisition time, i.e. the time of admission to time of acquiring infection, ranged from 3 to 31 days. All isolates were multiply resistant (MRAB), including resistance to carbapenems (MRAB-C) in the majority of cases but susceptible to tigecycline, with a minimum inhibitory concentration (MIC(90)) of 2 microg/mL. The overall mortality rate was 16.7%. Time-to-clearance of the MRAB-C was 8.3 days in the first outbreak, when tigecycline was not used, and 2.8 and 3.1 days during the second and third outbreaks, respectively, when tigecycline was used, and all but one patient survived. Environmental screening revealed gross contamination of many surfaces and equipment within the unit. The outbreak strains belonged to two distinct clones (D and E) whereas the 14 environmental strains belonged to three distinct groups (A-C). The outbreak of infections treated with tigecycline was successfully eliminated in conjunction with an aggressive infection control strategy.

The effect of hand hygiene compliance on hospital-acquired infections in an ICU setting in a Kuwaiti teaching hospital

Hand washing is widely accepted as the cornerstone of infection control in the intensive care unit (ICU). Nosocomial infections are frequently viewed as indicating poor compliance with hand washing guidelines. To determine the hand hygiene (HH) compliance rate among healthcare workers (HCWs) and its effect on the nosocomial infection rates in the ICU of our hospital, we conducted an interventional study. The study spanned a period of 7 months (February 2011-August 2011) and consisted of education about HH indications and techniques, workplace reminder posters, focused group sessions, and feedback on the HH compliance and infection rates. The WHO HH observation protocol was used both before and after a hospital-wide HH campaign directed at all staff members, particularly those in the ICU. Compliance was measured by direct observation of the HCWs, using observation record forms in a patient-directed manner, with no more than two patients observed simultaneously. The overall HH compliance rate was calculated by dividing the number of HH actions by the total number of HH opportunities. The nosocomial infection rates for the pre- and post-interventional periods were also compared to establish the effect of the intervention on rate of infections acquired within the unit. The overall rate of HH compliance by all the HCWs increased from 42.9% pre-intervention to 61.4% post-intervention, P<0.001. Individually, the compliance was highest among the nurses, 49.9 vs. 82.5%, respectively (P<0.001) and lowest among the doctors, 38.6 vs. 43.2%, respectively (P=0.24). The effect of the increase in the HH compliance rate on the nosocomial infection rate was remarkable. There were significant reductions in the following: the rate of overall health care-associated infections/1000 patient-days, which fell from 37.2 pre-intervention to 15.1 post-intervention (P<0.001); the rate of bloodstream infections, which fell from 18.6 to 3.4/1000 central-line-days (P<0.001); and the rate of lower respiratory tract infections, which fell from 17.6 to 5.2/1000 ventilator-days (P<0.001). Similarly, there were significant reductions in the isolation rates of 4 major hospital pathogens (P<0.001 and P=0.03). These findings suggest that although cross-infection in the ICU is a complex process, its frequency can be affected by meticulous adherence to hand hygiene recommendations.

Comparative evaluation of two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems for the identification of clinically significant yeasts

OBJECTIVES To prospectively evaluate the performance of two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry systems (MALDI-TOF MS) for the identification of clinically significant yeast isolates compared to the VITEK 2 system. METHODS One hundred and eighty-eight consecutive yeast isolates were analyzed by Bruker Biotyper and VITEK MS. The results were compared with the conventional VITEK 2 yeast identification system. Discrepant results were resolved by direct sequencing of rDNA. RESULTS Accurate identification by VITEK 2, VITEK MS, and Bruker Biotyper MS was 94.1% (177/188), 93.0% (175/188), and 92.6% (174/188), respectively. Three isolates were not identified by VITEK MS, while nine Candida orthopsilosis were misidentified as Candida parapsilosis, as this species is not present in its database. Eleven isolates were not identified or were wrongly identified by Bruker Biotyper and although another 14 were correctly identified, the score was unreliable at <1.7. CONCLUSION The overall accuracy of rapid MALDI-TOF MS systems was essentially comparable to that of the conventional VITEK 2 yeast identification system. However, future expansion of the databases may further improve the outcome and accuracy of identification of yeast species.

Evaluation of Curetis Unyvero, a Multiplex PCR-Based Testing System, for Rapid Detection of Bacteria and Antibiotic Resistance and Impact of the Assay on Management of Severe Nosocomial Pneumonia

ABSTRACT Health care-associated pneumonia due to multidrug-resistant organisms represents a major therapeutic challenge. Unfortunately, treatment is dependent on empirical therapy, which often leads to improper and inadequate antimicrobial therapy. A rapid multiplex PCR-based Unyvero pneumonia application (UPA) assay that assists in timely decision-making has recently become available. In this study, we evaluated the performance of UPA in detecting etiological pathogens and resistance markers in patients with nosocomial pneumonia (NP). The impact of this assay on the management of severe nosocomial pneumonia was also assessed. Appropriate specimens were processed by UPA according to the manufacturers protocol in parallel with conventional culture methods. Of the 56 patients recruited into the study, 49 (87.5%) were evaluable. Of these, 27 (55.1%) and 4 (8.2%) harbored multiple bacteria by the PCR assay and conventional culture, respectively. A single pathogen was detected in 8 (16.3%) and 4 (8.2%) patients, respectively. Thirteen different genes were detected from 38 patients, including the ermB gene (40.8%), the bla OXA-51-like gene (28.6%), the sul1 (28.6%) and int1 (20.4%) integrase genes, and the mecA and bla CTX-M genes (12.3% each). The time from sample testing to results was 4 h versus 48 to 96 h by UPA and culture, respectively. Initial empirical treatment was changed within 5 to 6 h in 33 (67.3%) patients based on the availability of UPA results. Thirty (62.2%) of the patients improved clinically. A total of 3 (6.1%) patients died, mainly from their comorbidities. These data demonstrate the potential of a multiplex PCR-based assay for accurate and timely detection of etiological agents of NP, multidrug-resistant (MDR) organisms, and resistance markers, which can guide clinicians in making early antibiotic adjustments.

Analysis of prevalence, risk factors and molecular epidemiology of Clostridium difficile infection in Kuwait over a 3-year period.

We conducted a prospective study to evaluate the prevalence and epidemiology of CDI in Kuwait government hospitals over a 3-year period, January 2003 to December 2005, to determine the ribotypes responsible for CDI and to estimate the prevalence of ribotype 027. We also conducted a case-control study to identify the risk factors in our patient population. A total of 697 stool samples from patients with suspected CDI were obtained and sent to Anaerobe Reference Laboratory, Faculty of Medicine, Kuwait University for Clostridium difficile toxin detection, culture and PCR ribotyping. During the period, 73 (10.5%) out of 697 patients met the case definition of CDI. Of these, 56 (76.7%) were hospital-acquired and 17 (23.3%) were from outpatient clinics. Thus, the prevalence of hospital-acquired CDI amongst patients with diarrhoea was 8% over the study period; the prevalence in 2003, 2004 and 2005 was 9.7%, 7.8% and 7.2%, respectively. Our data showed that 42.9% of the CDI patients were above 60 years, of which >79% were aged 71 years and above. Patients with CDI were more likely than the controls to have been exposed to immunosuppressive drugs and feeding via nasogastric tube. The most common ribotypes isolated during this study were 002, 001, 126 and 140 and they represent 55.1% of all isolates. PCR ribotype 027 was not isolated.