Cracking the Code: Unveiling the Diversity of Carbapenem-Resistant Klebsiella pneumoniae Clones in the Arabian Peninsula through Genomic Surveillance.

The rise of antimicrobial resistance is a global challenge that requires a coordinated effort to address. In this study, we examined the genetic similarity of carbapenem-resistant Klebsiella pneumoniae (CRKP) in countries belonging to the Gulf Cooperation Council (GCC) to gain a better understanding of how these bacteria are spreading and evolving in the region. We used in silico genomic tools to investigate the occurrence and prevalence of different types of carbapenemases and their relationship to specific sequence types (STs) of CRKP commonly found in the region. We analyzed 720 publicly available genomes of multi-drug resistant K. pneumoniae isolates collected from six GCC countries between 2011 and 2020. Our findings showed that ST-14 and ST-231 were the most common STs, and 51.7% of the isolates carried blaOXA-48-like genes. Additionally, we identified rare carbapenemase genes in a small number of isolates. We observed a clonal outbreak of ST-231 in Oman, and four Saudi isolates were found to have colistin resistance genes. Our study offers a comprehensive overview of the genetic diversity and resistance mechanisms of CRKP isolates in the GCC region that could aid in developing targeted interventions to combat this pressing global issue.

Elucidating the virulence genes harboured by carbapenemase- and non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae rectal isolates from patients admitted to intensive care units using whole-genome sequencing in Kuwait.

Introduction. Klebsiella pneumoniae is a Gram-negative pathogen responsible for community- and nosocomial-acquired infections. The presence of an accessory genome determines the bacterial pathogenicity and the host immune response, and thus indicates multidrug-resistant strains or more virulent groups. Little is known about the virulence genes in K. pneumoniae in Kuwait.Hypothesis/Gap Statement. The diversity of virulence genes and capsule loci in K. pneumoniae isolates warrants further genomic studies to better understand their transmission within the hospitals in Kuwait.Aim. We aimed to investigate the virulence genes harboured by K. pneumoniae isolated from rectal swabs of intensive care unit (ICU) patients in two Kuwaiti teaching hospitals.Methodology. Six isolates from patients in the ICUs of Al Razi and Mubarak hospitals, designated RZH144, RZH132 RZH108 and RZH173, and MKH381 and MKH347, respectively, were subjected to whole-genome sequencing (WGS) assays. RZH144 and RZH132 were non-carbapenemase-producing K. pneumoniae (NCKP) isolates negative for genes encoding carbapenemase production by PCR assays, and the remaining four were carbapenemase-producing K. pneumoniae (CPKP) isolates. Isolates were characterized by phenotypic, PCR and WGS methods. Susceptibility testing was performed by E test and clonality by multilocus sequence typing. Analysis of the isolates' assembled contigs was carried out using Kleborate (https://pathogen.watch).Results. An NCPE RZH132 K. pneumoniae isolate belonged to ST231-wzi104 and harboured gene clusters encoding the biosynthesis of the siderophore aerobactin (iuc5) on 62-3LV. The capsular locus variants were KL51 and O locus O1v2. Another NCPKP RZH144 isolate was confirmed as ST43-wzi412 and harboured KL61 and O1v1. The four CPKP isolates harboured two virulence loci - ybt14 and iuc5 - encoding the siderophores yersiniabactin and aerobactin, respectively. They belonged to ST231-wzi104 and harboured yersiniabactin on ICEKp5. The sequence type of ybt was YbST145-1LV. Strain RZ108 was devoid of virulence loci. Its sequence type was ST15-wzi151 and harboured KL48 and O1V1. ST231 clonal lineage isolates shared common virulence plasmid variants.Conclusion. The CPKP ST231 had the highest virulence score and contained iuc5, which was found for the first time in ST231-CPKP isolates in Kuwait.

Prevalence of carbapenem-resistant Enterobacteriaceae and emergence of high rectal colonization rates of blaOXA-181-positive isolates in patients admitted to two major hospital intensive care units in Kuwait.

BACKGROUND: Fecal colonization by carbapenem-resistant Enterobacteriaceae (CRE) can be the main reservoir for transmission of these resistant organisms especially in the Intensive Care Units (ICUs). AIM: This study was conducted to evaluate the rate of rectal carriage and molecular characterization of CRE in patients hospitalized in the ICUs of 2 major hospitals (Adan and Mubarak Al Kabeer Hospitals) in Kuwait. MATERIALS AND METHODS: Rectal swabs were collected from all patients at admission, 48 h after admission and once weekly from April 2017- March 2018. Initial CRE screening was carried out on MacConkey agar on which meropenem disc 10µg was placed. Identification of isolates was by API 20E. Susceptibility testing was performed using the E-test method. Polymerase chain reaction (PCR) was used to detect the carbapenemase-encoding genes. Clonal relationship was investigated by pulsed-field electrophoresis (PFGE). Genes of blaOXA-181 and blaNDM-5-carrying plasmids were detected in some strains. RESULTS: A total of 590 patients were recruited into the study. Of these, 58 were positive for CRE, giving a prevalence of 9.8%; 25/320 (7.8%) in Adan and 33/270 (12.2%) in Mubarak Al Kabeer Hospitals. All isolates were resistant to multiple antibiotics. Resistance rates to colistin and tigecycline were 17% and 83%, respectively. Single genes of blaOXA-181 were detected in isolates from 38 (65.5%) out of 58 patients and in 5 patients colonized by blaOXA-48-positive CRE. A combination of 2 genes was detected in 12 isolates; 5 blaKPC-2 and blaOXA-181, 4 blaVIM-1 and blaOXA-181, and 3 blaNDM-5 and blaOXA-181. PFGE showed an overall level of similarity of 38%. Southern hybridization studies localized the blaOXA-181 and blaNDM-5 genes to a large plasmid of 200kb in 3 K. pneumoniae isolates and a small plasmid of 80kb in 2 E. coli isolates, respectively. CONCLUSION: The prevalence of CRE colonization in the 2 hospital ICUs was relatively high and the emergence of blaOXA-181-mediated CRE is a cause for concern as there is the possibility of rapid horizontal spread among hospital patients in Kuwait. Active surveillance of CRE in the ICUs is highly recommended to stem its spread.

Surveillance of Antibiotic Resistance among Hospital- and Community-Acquired Toxigenic Clostridium difficile Isolates over 5-Year Period in Kuwait.

Clostridium difficile infection (CDI) is a leading and an important cause of diarrhea in a healthcare setting especially in industrialized countries. Community-associated CDI appears to add to the burden on healthcare setting problems. The aim of the study was to investigate the antimicrobial resistance of healthcare-associated and community-acquired C. difficile infection over 5 years (2008-2012) in Kuwait. A total of 111 hospital-acquired (HA-CD) and 35 community-acquired Clostridium difficile (CA-CD) clinical isolates from stool of patients with diarrhoea were studied. Antimicrobial susceptibility testing of 15 antimicrobial agents against these pathogens was performed using E test method. There was no evidence of resistance to amoxicillin-clavulanic acid, daptomycin, linezolid, piperacillin-tazobactam, teicoplanin and vancomycin by both HA-CD and CA-CD isolates. Metronidazole had excellent activity against CA-CD but there was a 2.9% resistance rate against HA-CD isolates. Ampicillin, clindamycin, levofloxacin and imipenem resistance rates among the HC-CD vs. CA-CD isolates were 100 vs. 47.4%; 43 vs. 47.4%; 100 vs. 100% and 100 vs. 89%, respectively. An unexpected high rifampicin resistance rate of 15.7% emerged amongst the HA-CD isolates. In conclusion, vancomycin resistance amongst the HA-CD and CA-CD isolates was not encountered in this series but few metronidazole resistant hospital isolates were isolated. High resistance rates of ampicillin, clindamycin, levofloxacin, and imipenem resistance were evident among both CA-CD and HA-CD isolates. Rifampicin resistance is emerging among the HA-CD isolates.

High Prevalence of New Delhi Metallo-ß-Lactamase-1 (NDM-1) Producers among Carbapenem-Resistant Enterobacteriaceae in Kuwait.

The aim of the study was to determine the prevalence of New Delhi metallo-ß lactamase-1 (NDM-1) producing Enterobacteriaceae in Kuwait over a one year period. Consecutive Enterobacteriaceae isolates with reduced susceptibility to carbapenems were collected from four government hospitals in Kuwait from January-December 2014. Their susceptibility to 18 antibiotics was performed by determining the minimum inhibitory concentration. Isolates resistant to carbapenems were tested by PCR for resistant genes. Finger printing of the positive isolates was done by DiversiLab®. Clinical data of patients harboring NDM-1 positive isolates were analyzed. A total of 764 clinically significant Enterobacteriaceae isolates were studied. Of these, 61 (8%) were carbapenem-resistant. Twenty one out of these 61 (34.4%) were NDM-1-producers. All patients positive for NDM-1-carrying bacteria were hospitalized. About half were females (11/21 [52.3%]), average age was 53.3 years and the majority were Kuwaitis (14/21 [66.6%]). Six patients (28.5%) gave a history of travel or healthcare contact in an endemic area. Mortality rate was relatively high (28.6%). The predominant organism was Klebsiella pneumoniae (14 [66.6%]) followed by E. coli (4 [19%]). All NDM-1-positive isolates were resistant to meropenem, ertapenem, cefotaxime, cefoxitin and ampicillin, while 95.2% were resistant to imipenem, cefepime, and piperacillin-tazobactam. They were multidrug resistant including resistance to tigecycline, but 90% remained susceptible to colistin. About two-thirds of isolates (61.9%) co-produced-extended spectrum ß-lactamases. During the study period, an outbreak of NDM-1 positive K. pneumoniae occurred in one hospital involving 3 patients confirmed by DiversiLab® analysis. In conclusion, NDM-1-producing Enterobacteriaceae is a growing healthcare problem with increasing prevalence in Kuwait, especially in hospitalized patients, leaving few therapeutic options. A high prevalence of NDM-1 necessitates the implementation of strict infection control to prevent the spread of these organisms.

Emergence of New Sequence Type OXA-48 Carbapenemase-Producing Enterobacteriaceae in Kuwait.

The aim of this study was to investigate the infections due to OXA-48 carbapenemase-producing bacteria in tertiary hospitals in Kuwait (September 2011 to April 2013) and to determine the sequence types (STs) of the corresponding isolates. Eleven OXA-48 carbapenemase-producing Enterobacteriaceae isolates were recovered from patients treated in nine different hospitals in Kuwait. Susceptibility testing to eighteen antibiotics was done using the E-test. PCR assays were performed for the detection of genes encoding extended-spectrum ß-lactamases (ESBLs) (blaCTX-M, blaSHV, and blaTEM) and carbapenemases (blaOXA-48, blaVIM, blaNDM, blaIMP, blaGIM, and blaKPC). STs were determined by Multilocus Sequence Typing. Seven Klebsiella pneumoniae, two Escherichia coli, one Enterobacter cloacae, and one Morganella morganii harbored the blaOXA-48 gene. The K. pneumoniae and E. coli belonged to seven and two different STs, respectively, which were not related to those reported from this region. The majority of the isolates carried either blaCTX-M or blaSHV genes and showed a multidrug-resistant phenotype, including resistance to tigecycline. Multidrug-resistant Enterobacteriaceae harboring the blaOXA-48 gene are emerging in Kuwait with different STs compared to those identified in other countries of the region. Detection of OXA-48-producing Enterobacteriaceae in Kuwait is important to prevent its rapid spread.

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.

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.

A study of the bacterial flora before and after antiseptic skin preparation of the perineum in male urology patients.

OBJECTIVE: To establish the bacterial flora of the perineum before and after antiseptic preparation in male urology patients. PATIENTS/METHODS: Adult male patients undergoing cystoscopic procedures were studied. Three sets of swab specimens, labelled A, B and C, were taken from the perineum in the theatre. Specimen A was taken before cleaning and disinfection of the skin with Savlon (Chlorhexidine- cetrimide mixture), specimen B after disinfection and draping, and C after completion of the operative procedure. Specimens were processed on standard laboratory media for aerobic and anaerobic bacteria and yeasts. RESULTS: Of the 114 patients studied, 43 (37.7%) had a positive culture for significant microorganisms in specimen A, 7 (6.1%) in specimen B and 13 (11.4%) in specimen C (A vs. B p < 0.001, A vs. C p < 0.001, B vs. C p < 0.01). The commonest isolates in specimen A were Gram-positive organisms (84.1%). The positive-culture rate for patients with end-stage renal failure was 71.4%, for those with a urethral catheter it was 53.8%, for those without systemic diseases it was 36.6% and for patients with diabetic mellitus it was 28.1%. CONCLUSION: About 38% of patients undergoing cystoscopic procedures had a significant positive perineal culture, with Staphylococcus species being the predominant skin flora. The bacteria culture rate was affected by the presence of systemic diseases. The use of Savlon to disinfect the perineum resulted in a significant reduction in the bacterial count of the perineum.

Molecular characterization of nitroimidazole resistance in metronidazole-resistant bacteroides species isolated from hospital patients in Kuwait.

OBJECTIVES: The aim of this study was to screen for infections caused by metronidazole (MTZ)-resistant Bacteroides spp., and to characterize the genes that encode the MTZ resistance. MATERIALS AND METHODS: A total of 7 MTZ-resistant Bacteroides spp. were isolated from 5 patients with MTZ-resistant infections. These organisms were investigated for carriage of genes that encode MTZ resistance. The presence of these genes was investigated by PCR and the PCR products were subjected to PCR-RFLP analysis. RESULTS: The strains were MTZ-resistant with minimum inhibitory concentrations of > 32 microg/ml. The presence of nim genes was indicated by PCR in all 7 strains. PCR-RFLP analysis of the nim gene products demonstrated two of the five reported resistance genes, nimA-nimE. These two resistance genes were nimE in 5 of the 7 isolates and nimA in 2 strains. CONCLUSION: MTZ-resistant Bacteroides spp. have been isolated from patients in Kuwait. Nim genes, specifically nimE and nimA, mediate the drug resistance in these isolates. The methods used in detecting these genes are rapid, accurate and relatively inexpensive and could be adopted easily to help in monitoring emergence of MTZ resistance determinants in Kuwait.

Prevalent PCR ribotypes of clinical and environmental strains of Clostridium difficile isolated from intensive-therapy unit patients in Kuwait.

Ninety-five isolates of Clostridium difficile from symptomatic and asymptomatic patients and 18 from their environment in the intensive-therapy units (ITUs) of four teaching hospitals in Kuwait were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping). A total of 32 different ribotypes was detected among the clinical isolates. The predominant ribotypes from the clinical isolates were types 097 and 078, which accounted for approximately 40 % of all isolates in the ITUs in Kuwait. Ribotypes 097 (toxigenic), 078 (toxigenic) and 039 (non-toxigenic) were three distinct clones that were circulating in all four hospitals. Ribotypes 097, 078 and 076 (i.e. 50 % of isolates from symptomatic patients) were the predominant isolates associated with C. difficile-associated disease (CDAD). The environmental isolates belonged to a diverse range of ribotypes, with no particular types common to all the hospitals. Ribotype 078 was found only in the patient environment in Mubarak hospital, while ribotype 097 was restricted to Amiri hospital. The hospital environment occupied by symptomatic as well as symptom-free patients was contaminated with C. difficile. Eight new strains that did not match any in the PCR ribotype library established at the PHLS Anaerobe Reference Unit, Cardiff, UK, were assigned ribotypes 105, 125, 128, 129, 131, 134, 140 and 141. These findings show that the isolates associated with CDAD in Kuwait are different from those found in the UK and some other European countries.

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.