Molecular Screening of Carbapenem-Resistant K. pneumoniae (CRKP) Clinical Isolates for Concomitant Occurrence of Beta-Lactam Genes (CTX-M, TEM, and SHV) in the Kingdom of Bahrain.

The emergence of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae, including CRKP infections, has resulted in significant morbidity and mortality worldwide. We aimed to explore the presence of bla genes (CTX-M, TEM, and SHV) in CRKP isolates. A total of 24 CRKP isolates were randomly selected from the Salmaniya Medical Complex Microbiology Laboratory. These isolates, which were positive for carbapenemases, were further explored for CTX-M, TEM, and SHV genes using PCR. All the CTX-M PCR amplicons were sent for sequencing. To determine genetic relatedness, molecular typing by ERIC-PCR was performed. The bla gene testing demonstrated that a significant proportion of these isolates harbored SHV, CTX-M, and TEM genes (100%, 91.6%, and 45.8%), respectively. Bioinformatic analyses confirmed CTX-M-15 in these isolates. ERIC-PCR analysis showed three clusters demonstrating genetic relatedness. The study findings reveal the concomitant carriage of the SHV and CTX-M-15 and a comparatively lower carriage of TEM genes in CRKP isolates. Our findings highlight the significance of routinely reporting the presence of antibiotic resistance genes along with regular antibiotic sensitivity reports, as this will aid clinicians in prescribing appropriate antibiotics.

Reduction of Panton-Valentine Leukocidin Production in the Staphylococcal Strain USA300 After In Vitro Ascorbic Acid and Nicotinamide Treatment.

Background Panton-Valentine leukocidin (PVL) is one of the most important determinants of virulence in Staphylococcus aureus. It is associated with a propensity for complicating skin and soft tissue infections and necrotizing pneumonia. This study aims to quantitively examine the effect of ascorbic acid and nicotinamide on PVL production in the reference strain USA300. Methodology Sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantitively measure the production of PVL via the commercial LukS sandwich ELISA kit (IBT Bio-services, MD, USA). Results Incubating USA300 with subinhibitory concentrations of antioxidants resulted in a statistically significant eight-fold reduction in PVL production at 1.25 mg/mL and 30 mg/mL for ascorbic acid and nicotinamide, respectively. Although the mechanism by which antioxidants inhibit PVL production is yet to be elucidated, we suggest that it can be due to interrupting PVL gene expression. Conclusions Ascorbic acid and nicotinamide have the potential to be toxin-suppressing agents that may be effective in supporting the bactericidal effect of antibiotics to improve the outcome of PVL-associated infections; however, further extensive research is required.

Prevalence of Carbapenemases in Carbapenem-Resistant Acinetobacter baumannii Isolates from the Kingdom of Bahrain.

BACKGROUND: Acinetobacter baumannii is regarded as a significant cause of death in hospitals. The WHO recently added carbapenem-resistant Acinetobacter baumannii (CRAB) to its global pathogen priority list. There is a dearth of information on CRAB from our region. METHODS: Fifty CRAB isolates were collected from four main hospitals in Bahrain for this study. Bacterial identification and antibiotic susceptibility tests were carried out using the BD PhoenixTM and VITEK-2 compact, respectively. Using conventional PCR, these isolates were further screened for carbapenem resistance markers (blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-40, blaIMP, blaNDM, blaVIM, and blaKPC). RESULTS: All of the isolates were resistant to imipenem (100%), meropenem (98%), and cephalosporins (96-98%), followed by other commonly used antibiotics. All these isolates were least resistant to gentamicin (64%). The detection of resistance determinants showed that the majority harbored blaOXA-51 (100%) and blaIMP (94%), followed by blaOXA-23 (82%), blaOXA-24 (46%), blaOXA-40 (14%), blaNDM (6%), blaVIM (2%), and blaKPC (2%). CONCLUSION: The study isolates showed a high level of antibiotic resistance. Class D carbapenemases were more prevalent in our CRAB isolate collection. The resistance genes were found in various combinations. This study emphasizes the importance of strengthening surveillance and stringent infection control measures in clinical settings to prevent the emergence and further spread of such isolates.

Multidrug-Resistant Staphylococcus aureus Isolates in a Tertiary Care Hospital, Kingdom of Bahrain.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a ubiquitous pathogen associated with a wide spectrum of human infections. In recent decades, MRSA infections have been increasingly reported in individuals without established risk factors, infecting immunocompetent members of the community. This emergence is attributed to the production of various virulence factors, notably Panton-Valentine leukocidin (PVL). OBJECTIVE: The aim of this study was to better understand the prevalence, antibiotic resistance profiles, and molecular characteristics of S. aureus and MRSA in a tertiary care hospital in the Kingdom of Bahrain. MATERIALS AND METHODS: This cross-sectional study was carried out in a tertiary hospital for a one-year period, from December 2020 to December 2021. A total of 161 consecutive S. aureus isolates were collected. Antibiotic susceptibility was tested using BD Phoenix™ automated identification and susceptibility testing system. Molecular analysis was conducted via conventional PCR and conventional multiplex PCR for SCCmec typing. RESULTS: In this study, 161 S. aureus isolates were investigated, 60% (n=97) were characterized as MRSA, of which, 12% (n=12) were healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) while 88% (n=85) were community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). No statistically significant difference (P>0.05) in antibiotic resistance trends between HA-MRSA and CA-MRSA was detected. Multidrug resistance (MDR) amounted to 19% (n=30) of all S. aureus isolates, 14% (n=9) of methicillin-susceptible Staphylococcus aureus (MSSA) isolates, and 22% (n=21) of MRSA isolates. SCCmec typing demonstrated a high prevalence of type IV (61%, n=59), followed by type V (32%, n=31), then type II (4%, n=4), and type III (3%, n=3). The PVL prevalence was 39% (n=25) in MSSA and 62% (n=60) in MRSA, 33% (n=4) in HA-MRSA, and 66% (n=56) in CA-MRSA. CONCLUSION: This study demonstrated the emergence of PVL-producing CA-MRSA in a tertiary care hospital, as well as the detection of PVL-producing MDR strains. This development prompts serious measures to be taken in order to sustain a healthy clinical environment.

Clinical carbapenem-resistant Klebsiella pneumoniae isolates simultaneously harboring bla NDM-1, bla OXA types and qnrS genes from the Kingdom of Bahrain: Resistance profile and genetic environment.

The prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) is currently increasing worldwide, prompting WHO to classify it as an urgent public health threat. CRKP is considered a difficult to treat organism owing to limited therapeutic options. In this study, a total of 24 CRKP clinical isolates were randomly collected from Salmaniya Medical Complex, Bahrain. Bacterial identification and antibiotic susceptibility testing were performed, on MALDI-TOF and VITEK-2 compact, respectively. The isolates were screened for carbapenem resistance markers (bla NDM, bla OXA-23, bla OXA-48 and bla OXA-51) and plasmid-mediated quinolone resistance genes (qnrA, qnrB, and qnrS) by monoplex PCR. On the other hand, only colistin-resistant isolates (n=12) were screened for MCR-1, MCR-2 and MCR-3 genes by monoplex PCR. Moreover, the Genetic environment of bla NDM, integrons analysis, and molecular characterization of plasmids was also performed. Antibiotic susceptibility revealed that all the isolates (100%) were resistant to ceftolozane/tazobactam, piperacillin/tazobactam, 96% resistant to ceftazidime, trimethoprim/sulfamethoxazole, 92% resistant to meropenem, gentamicin and cefepime, 88% resistant to ciprofloxacin, imipenem, and 37% resistant to amikacin. Ceftazidime/avibactam showed the least resistance (12%). 75% (n=12/16) were resistant to colistin and 44% (n=7/16) showed intermediate susceptibility to tigecycline. The detection of resistant determinants showed that the majority (95.8%) of CRKP harbored bla NDM-1, followed by bla OXA-48 (91.6%) bla OXA-51 (45.8%), and bla OXA-23 (41.6%). Sequencing of the bla NDM amplicons revealed the presence of bla NDM-1. Alarmingly, 100% of isolates showed the presence of qnrS. These predominant genes were distributed in various combinations wherein the majority were bla NDM-1 + bla OXA-51+ qnrS + bla OXA-48 (n =10, 41.7%), bla NDM-1 + bla OXA-23+ qnrS + bla OXA-48 (n=8, 33.3%), among others. In conclusion, the resistance rate to most antibiotics is very high in our region, including colistin and tigecycline, and the genetic environment of CRKP is complex with the carriage of multiple resistance markers. Resistance to ceftazidime/avibactam is uncommon and hence can be used as a valuable option for empirical therapy. Molecular data on resistance markers and the genetic environment of CRKP is lacking from this geographical region; this would be the first report addressing the subject matter. Surveillance and strict infection control strategies should be reinforced in clinical settings to curb the emergence and spread of such isolates.

Reported Adverse Effects and Attitudes among Arab Populations Following COVID-19 Vaccination: A Large-Scale Multinational Study Implementing Machine Learning Tools in Predicting Post-Vaccination Adverse Effects Based on Predisposing Factors.

Background: The unprecedented global spread of coronavirus disease 2019 (COVID-19) has imposed huge challenges on the healthcare facilities, and impacted every aspect of life. This has led to the development of several vaccines against COVID-19 within one year. This study aimed to assess the attitudes and the side effects among Arab communities after receiving a COVID-19 vaccine and use of machine learning (ML) tools to predict post-vaccination side effects based on predisposing factors. Methods: An online-based multinational survey was carried out via social media platforms from 14 June to 31 August 2021, targeting individuals who received at least one dose of a COVID-19 vaccine from 22 Arab countries. Descriptive statistics, correlation, and chi-square tests were used to analyze the data. Moreover, extensive ML tools were utilized to predict 30 post vaccination adverse effects and their severity based on 15 predisposing factors. The importance of distinct predisposing factors in predicting particular side effects was determined using global feature importance employing gradient boost as AutoML. Results: A total of 10,064 participants from 19 Arab countries were included in this study. Around 56% were female and 59% were aged from 20 to 39 years old. A high rate of vaccine hesitancy (51%) was reported among participants. Almost 88% of the participants were vaccinated with one of three COVID-19 vaccines, including Pfizer-BioNTech (52.8%), AstraZeneca (20.7%), and Sinopharm (14.2%). About 72% of participants experienced post-vaccination side effects. This study reports statistically significant associations (p < 0.01) between various predisposing factors and post-vaccinations side effects. In terms of predicting post-vaccination side effects, gradient boost, random forest, and XGBoost outperformed other ML methods. The most important predisposing factors for predicting certain side effects (i.e., tiredness, fever, headache, injection site pain and swelling, myalgia, and sleepiness and laziness) were revealed to be the number of doses, gender, type of vaccine, age, and hesitancy to receive a COVID-19 vaccine. Conclusions: The reported side effects following COVID-19 vaccination among Arab populations are usually non-life-threatening; flu-like symptoms and injection site pain. Certain predisposing factors have greater weight and importance as input data in predicting post-vaccination side effects. Based on the most significant input data, ML can also be used to predict these side effects; people with certain predicted side effects may require additional medical attention, or possibly hospitalization.

Dysregulation of human beta-defensin-3 expression in the peripheral blood of patients with sepsis.

OBJECTIVES: Sepsis is a serious medical condition caused by the body's systemic inflammatory response to infections. The antimicrobial peptides, human beta-defensins, play a key role in modulating host immune responses, and aberrant expression of human beta-defensins has been implicated in many infections and inflammatory diseases. However, little is known about the expression of human beta-defensin-3 in systemic infectious diseases. METHODS: We investigated the gene expression and protein level of human beta-defensin-3 in peripheral whole blood from 107 participants-67 patients with sepsis and 40 healthy controls-and evaluated the feasibility of human beta-defensin-3 as an indicator for sepsis. Total RNA was extracted from peripheral blood samples, and relative mRNA expression of human beta-defensin-3 was determined by reverse transcription-quantitative polymerase chain reaction. Plasma concentration of human beta-defensin-3 was measured by enzyme-linked immunosorbent assay. Pearson's correlation analysis was performed to assess the relationship between human beta-defensin-3 mRNA and protein levels. Receiver operating characteristic analysis was performed to evaluate the value of human beta-defensin-3 as a biomarker for sepsis. RESULTS: Human beta-defensin-3 mRNA expression was significantly downregulated in sepsis patients compared to controls (p = 0.001). The mean fold change of mRNA expression (±standard error) was 0.82 ± 0.63 in sepsis patients and 1.39 ± 1.09 in controls. Plasma concentration of human beta-defensin-3 (pg/mL) was significantly lower in sepsis patients compared to healthy controls (p = 0.039). The mean protein concentration (±standard error) was 539.6 ± 39.4 in sepsis patients and 715.5 ± 53 in controls. There was a significant correlation between human beta-defensin-3 mRNA expression and the corresponding protein level in sepsis patients (r = 0.358, p = 0.04), but not in healthy controls (r = 0.124, p = 0.51). For discriminating sepsis patients from healthy controls, the area under the receiver operating characteristic curve was 0.722 (95% confidence interval: 0.597-0.847, p = 0.002) for human beta-defensin-3 mRNA and 0.689 (95% confidence interval: 0.557-0.827, p = 0.009) for human beta-defensin-3 protein. CONCLUSION: This is the first study to show the downregulation of human beta-defensin-3 gene expression and protein level in sepsis, which may contribute to the complex immunological imbalance in sepsis. The significant correlation between human beta-defensin-3 mRNA expression and protein concentration suggests that mRNA expression could be used to predict protein level. Our study also showed a potential role of human beta-defensin-3 as a blood-based biomarker for sepsis. More studies on the clinical significance of human beta-defensin-3 in sepsis could further support a biomarker development.

Outcomes of COVID-19 in the Eastern Mediterranean Region in the first 4 months of the pandemic.

OBJECTIVES: To summarize the outcomes of the coronavirus disease 2019 infections in the Eastern Mediterranean Region (EMR) in the first 4 months of the pandemic. METHODS: A meta-analysis approach was used in this context. We used the aggregate data from the World Health Organization Regional Office for the EMRO (until 26 May 2020) to generate this report. RESULTS: An analysis of official data from all 22 countries and territories in the Middle East, North Africa, the Horn of Africa, and Central Asia K=22 (a total of 438,717 cases) was performed. The total number of cases, recovered cases were 438,717,228,986,  and deaths was 11,290 in the EMR. Meta-analytic pooling of the point estimates of recovery rate per country in the EMR was 52.5% (95% CI 52.3% - 52.6%). The lowest recovery rates were in Somalia (4.3%), and the highest rates were in Tunisia (87.4%). Meta-analytic pooling of the point estimates of death rate per country in the EMR yielded 3.85% [95% CI 3.80% - 3.9%]. Meta-analytic pooling of the point estimates of recovery rate per country in the Gulf Cooperation Council countries was 46.1% (95% CI 45.8% - 46.3%). Meta-analytic pooling of the point estimates of death rate per country in the Gulf Cooperation Council countries was 0.6% (95% CI 0.50% - 0.65%). CONCLUSION: Wide variability was found between EMR countries in recovery and mortality, implying the possible impact of resource availability, and genetic and environmental factors on the morality and recovery of the COVID-19.

Exploring the evidence base for national and regional policy interventions to combat resistance.

The effectiveness of existing policies to control antimicrobial resistance is not yet fully understood. A strengthened evidence base is needed to inform effective policy interventions across countries with different income levels and the human health and animal sectors. We examine three policy domains-responsible use, surveillance, and infection prevention and control-and consider which will be the most effective at national and regional levels. Many complexities exist in the implementation of such policies across sectors and in varying political and regulatory environments. Therefore, we make recommendations for policy action, calling for comprehensive policy assessments, using standardised frameworks, of cost-effectiveness and generalisability. Such assessments are especially important in low-income and middle-income countries, and in the animal and environmental sectors. We also advocate a One Health approach that will enable the development of sensitive policies, accommodating the needs of each sector involved, and addressing concerns of specific countries and regions.

High prevalence of blaCTX-M in Enterobacteriaceae isolates from the Kingdom of Bahrain.

OBJECTIVE: To determine the molecular epidemiology of extended-spectrum ß-lactamase (ESBL) by testing a cohort of clinical ESBL-producing bacterial isolates that were isolated in the Kingdom of Bahrain. METHODS: ESBL producing Enterobacteriaceae isolates (based on phenotypic tests) were collected from Microbiology Laboratory of the Salmaniya Medical Complex, Bahrain between January-June 2006. Antibiotic susceptibility to a panel of antibiotics was performed and bla(CTX-M) genes were detected by multiplex PCR. RESULTS: A total of 230 isolates (Escherichia coli, n=180; Klebsiella pneumoniae, n=50) were studied, 98% were CTX-M type. For Escherichia coli isolates, 65 (36.1%) harbored CTXM+TEM combination and 68 (37.8%) had CTX-M alone. In contrast, for Klebsiella pneumoniae isolates only 5 (10.0%) harbored the CTX-M combination, and none had CTX-M only. The bla(CTX-M) gene was found predominantly in urine isolates (n=145/230; 63.0%). Sensitivity to imipenem and nitrofurantoin was 100% and 60%, respectively. CTX-M carriage was associated with the resistance to fluoroquinolones, trimethoprim-sulfamethoxazole and aminoglycosides. CONCLUSIONS: Our study documentes high prevalence of CTX-M ESBL type among Escherichia coli and Klebsiella from the Kingdom of Bahrain. The apparent dissemination of CTX-M producers could represent a substantial barrier in the treatment of community-acquired infections. The use of extended-spectrum cephalosporins, quinolones, and aminoglycosides is compromised, leaving carbapenems as the therapeutic option for severe infections caused by ESBL producers.

Extended spectrum beta-lactamases (ESBL) in Escherichia coli and Klebsiella pneumoniae: trends in the hospital and community settings.

BACKGROUND: To assess the prevalence of extended spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella strains in nosocomial and community-acquired infections. METHODOLOGY: The study was conducted at a centralized microbiology laboratory in the Eastern Province of Saudi Arabia. Laboratory records (January 2004 - December 2005) were assessed. Associated resistance to a panel of antibiotics was determined. RESULTS: A total of 6,750 Gram-negative organisms were assessed for ESBL-phenotype. ESBL was detected in 6% (409/6,750) of isolates, the majority of which were E. coli (83%). ESBL producers were significantly higher among isolates from in-patients 15.4% (143/927) versus out-patients (4.5%; 266/5,823); p < 0.05. Old age (older than 60 years) represented a significant risk for having an ESBL-producing pathogen. Urine was the major source of ESBL isolates in in-patients (46.1%) and out-patients (74.4%). The proportion of urinary E. coli isolates which were ESBL producers was significantly higher among in-patients (53/506; 10.4%) compared to out-patients (182/4,074; 4.4%); p<0.05. Old age (older than 60 years) represented a significant risk for having an ESBL-producing pathogen. Urine was the major source of ESBL isolates in in-patients (46.1%) and out-patients (74.4%). The proportion of urinary E. coli isolates which were ESBL producers was significantly higher among in-patients (53/506; 10.4%) compared to out-patients (182/4,074; 4.4%); p<0.05. Among in-patients, 60% of the ESBL associated infections were nosocomial. All were sensitive to imipenem but high levels of resistance to gentamicin, amikacin, amoxicillin-clavulanic acid and ciprofloxacin was shown. CONCLUSION: The findings document evidence of the spread of multiresistant ESBL-producers into the community. This has significant implications for patient management, and indicates the need for increased surveillance and molecular characterization of these isolates.

Clinical relevance of virulence genes in Campylobacter jejuni isolates in Bahrain.

There are no data describing the genetic make-up of Campylobacter strains (an important aetiological agent of diarrhoea) circulating in the Arabian Gulf region. Here, the molecular characterization of two virulence genes in Campylobacter jejuni from Bahrain and the relationship with clinical infection are reported. Molecular screening for cytolethal distending toxin (cdtB) and invasion-associated marker (iam) genes was carried out on C. jejuni stool isolates collected from January 2002 to January 2004 in Bahrain. The molecular characterization was correlated with the patients' socio-demographic and clinical parameters. Of the 96 C. jejuni strains tested, 50 (52 %) were cdtB+/iam+, 30 (31 %) were cdtB+/iam- and 16 (17 %) were cdtB-/iam-. Sixty-nine per cent (66/96) of patients were less than 3 years old, with significantly higher detection of cdtB+/iam+ and cdtB+/iam- strains (P < 0.001 and P < 0.01, respectively) in this age group. Seventy patients (73 %) were symptomatic. In the group that were less than 3 years old, 62 and 85 % of those with cdtB+/iam+ and cdtB+/iam- strains, respectively, were symptomatic compared with 100 % for those over 3 years of age. However, the presence of cdtB-/iam- strains still resulted in clinical infection in the children under 3 years but not in the older patients. This is the first report describing the molecular characterization of virulence genes in Campylobacter isolates from this region. The findings indicate that strains of different virulence genetic make-up are circulating in the population, with children under the age of 3 years being most vulnerable. Further work on the molecular characterization, gene expression and determination of the invasive phenotypes of C. jejuni strains circulating in different regions is needed.

Neonatal sepsis 1991-2001: prevalent bacterial agents and antimicrobial susceptibilities in Bahrain.

OBJECTIVES: To investigate the organisms causing neonatal sepsis and their modifications over an extended period, to assess their changing sensitivities to antibiotics and to verify whether the policy for screening pregnant women for group B streptococci (GBS) carriage is desirable in our settings. SUBJECTS AND METHODS: Medical records of all infants with positive blood culture from the Neonatal Intensive Care Unit at Salmaniya Medical Complex between 1991 and 2001 and Bahrain Defense Force Hospital between 1999 and 2001 were reviewed. RESULTS: Of the 7,978 neonates in both hospitals 335 (4.19%) had culture-proven bacteremia. Gram-positive bacteria were isolated at constant rate over the 11-year period. The main agents isolated were coagulase-negative Staphylococcus (CoNS) in 138 cases (41%), Staphylococcus aureus in 28 newborns (8%) and GBS in 26 patients (7.8%, 0.2/1,000 live births). All of them were sensitive to penicillin G, erythromycin and clindamycin. Gram-negative bacteria were declining but Escherichia coli was isolated in 35 cases (10%). Of special concern is the increasing percentage (5.7%) of Candida isolation. No clear trend toward increasing resistance was observed, although a major difference among the two institutions was evident. Klebsiella and Enterobacter spp. showed resistance to many of the antibiotics tested, thereby posing difficult therapeutic choices. CONCLUSION: Good quality specimens are essential to evaluate the role of CoNS. The increasing threat of fungal infection must be carefully tackled. Specifically tailored policies for GBS prevention must be defined according to the local epidemiology.

Effect of antibiotic sub inhibitory concentration on cytolethal distending toxin production by Campylobacter jejuni.

OBJECTIVES: To investigate the effect of pre-exposure antibiotics on cytolethal distending toxin (CDT) production and toxigenic effect of C. jejuni. METHODS: Sonicates and filtrates were prepared from known cdt+ and cdt- isolates of C. jejuni which had been pre-exposed to varying concentrations (MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC) of erythromycin and ciprofloxacin. The CDT toxigenic effect was examined using INT 407 and HeLa cells. RESULTS: A trend of increased toxigenic effect was observed with pre-exposure to antibiotics. This was more pronounced with erythromycin pre-exposure compared to ciprofloxacin. Although a trend of increasing toxigenic effect with decreasing antibiotic concentration was demonstrable, some differences were observed between isolates. In one isolate the increased toxigenic effect was statistically significant (P<0.05) at 1/4 MIC in INT 407 cells and at 1/8 MIC in HeLa cells. CONCLUSIONS: This study provides evidence of an association between CDT production by C. jejuni and pre-exposure to antibiotics. Pre-exposure to ciprofloxacin and erythromycin at concentrations below MICs could potentiate CDT activity. Further work is needed to elucidate the mechanism involved. We recommend that these antibiotics be used in the treatment of C. jejuni enteritis only when strongly indicated and with careful monitoring of patients.