Meningitis and Bacteremia by Unusual Serotype of Salmonella enterica Strain: A Whole Genome Analysis.

Background: Although meningitis caused by Salmonella species is relatively rare and accounts for <1% of the confirmed cases in neonates, it is associated with case complications and fatality rates up to 50-70% when compared to other forms of Gram-negative bacilli meningitis. Objectives: We conducted an investigation into the first reported case of neonatal meningitis caused by nontyphoidal S. enterica in Jazan, a region in the southwestern part of Saudi Arabia. Methods: CSF and blood culture were collected from a female neonate patient to confirm the presence of bacterial meningitis. WGS was conducted to find out the comprehensive genomic characterization of S. enterica isolate. Results: A 3-week-old infant was admitted to a local hospital with fever, poor feeding, and hypoactivity. She was diagnosed with Salmonella meningitis and bacteremia caused by S. enterica, which was sensitive to all antimicrobials tested. WGS revealed the specific strain to be S. enterica serotype Johannesburg JZ01, belonging to ST515 and cgMLST 304742. Conclusions: We presented a genomic report of rare case of NTS meningitis in an infant who is living in a rural town in Jazan region, Saudi Arabia. Further research is required to understand the impact of host genetic factors on invasive nontyphoidal Salmonella infection.

Genomic analysis of extensively drug resistant (XDR) Klebsiella pneumoniae high-risk clone ST14 co-harboring blaNDM and blaOXA-48 recovered from Saudi Arabia.

BACKGROUND: This study presents a comprehensive genomic analysis of NDM and OXA-48-producing Klebsiella pneumoniae in the Western region of Saudi Arabia, traversed by tens of millions of Muslims from various countries annually. This significant influx of visitors invariably leads to the spread and diversity of MDR bacteria. METHODS: Genome sequencing was performed using MiSeq system of 29 CPKP isolates that were NDM and OXA-48-positive isolated from nosocomial infections and demonstrated resistance to most antibiotics, including carbapenems. RESULTS: WGS analysis showed that 12 (41.3%) isolates co-harbored blaOXA-48,blaCTX-M-15 and blaNDM genes. Notably, 16 (55.1%) isolates were identified as high-risk clone ST14, with 50% of these isolates co-harbored blaOXA-48, blaNDM and blaCTX-M-15 genes. All ST14 isolates were identified as capsular genotype KL2 and O1/O2v1 antigen with yersiniabactin locus ypt 14 carried by ICEKp5. The two isolates were identified as ST2096/KL64 hypervirulent K. pneumoniae (hvKp) clone harboring several virulence factors, including the regulator of the mucoid phenotype rmpA2 and aerobactin (iuc-1). Interestingly, two of the hvKp ST383/KL30 isolates were resistant to all tested antimicrobials except colistin and tigecycline, and simultaneously carried numerous ESBLs and carbapenemase genes. These isolates also harbor several virulence factors such as rmpA1, rmpA2, carried on KpVP-1, and aerobactin (iuc-1). CONCLUSION: this study provides insights into the spread and prevalence of high-risk clones of CPKP in the Western region of Saudi Arabia. The ST14 high-risk clone appears to be the predominant CPKP clone in this region, posing a significant threat to public health. This study also reports the presence of two globally disseminated hypervirulent K. pneumoniae (hvKp) clones, namely ST2096 and ST383. Therefore, it is essential to improve surveillance and implement strict infection control measures in this region, which receives a substantial number of visitors to effectively monitor and reduce the spread of high-risk clones of antimicrobial-resistant bacteria, including CPKP.

Microbiological risk assessment and resistome analysis from shotgun metagenomics of bovine colostrum microbiome.

Colostrum is known for its nutraceutical qualities, probiotic attributes, and health benefits. The aim of this study was to profile colostrum microbiome from bovine in rural sites of a developing country. The focus was on microbiological safety assessments and antimicrobial resistance, taking into account the risks linked with the consumption of raw colostrum. Shotgun sequencing was employed to analyze microbiome in raw buffalo and cow colostrum. Alpha and beta diversity analyses revealed increased inter and intra-variability within colostrum samples' microbiome from both livestock species. The colostrum microbiome was mainly comprised of bacteria, with over 90% abundance, whereas fungi and viruses were found in minor abundance. Known probiotic species, such as Leuconostoc mesenteroides, Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus paracasei, were found in the colostrum samples. A relatively higher number of pathogenic and opportunistic pathogenic bacteria were identified in colostrum from both animals, including clinically significant bacteria like Clostridium botulinum, Pseudomonas aeruginosa, Escherichia coli, and Listeria monocytogenes. Binning retrieved 11 high-quality metagenome-assembled genomes (MAGs), with three MAGs potentially representing novel species from the genera Psychrobacter and Pantoea. Notably, 175 antimicrobial resistance genes (ARGs) and variants were detected, with 55 of them common to both buffalo and cow colostrum metagenomes. These ARGs confer resistance against aminoglycoside, fluoroquinolone, tetracycline, sulfonamide, and peptide antibiotics. In conclusion, this study describes a thorough overview of microbial communities in buffalo and cow colostrum samples. It emphasizes the importance of hygienic processing and pasteurization in minimizing the potential transmission of harmful microorganisms linked to the consumption of colostrum.

Prevalence and molecular determinants of carbapenemase-producing Klebsiella pneumoniae isolated from Jazan, Saudi Arabia.

INTRODUCTION: The World Health Organization (WHO) designated Carbapenem-resistant Enterobacterales (CRE), formerly Enterobacteriaceae, among the global priority list of antibiotic-resistant bacteria. The rate of CRE in Arabian countries, including Saudi Arabia has increased. Here, we report the prevalence of carbapenemase-producing Klebsiella pneumoniae (CPKP) in the Jazan region, a southern coastal province of Saudi Arabia. METHODOLOGY: Eighty-six non-repetitive clinical isolates of K. pneumoniae that showed resistance to at least one of the carbapenem drugs were collected from three tertiary hospitals in the Jazan region from March 2020 to April 2021. The identification and antimicrobial susceptibility testing (AST) of isolates were performed using various automated systems. Molecular detection of carbapenemase genes was conducted using a multiplex PCR. RESULTS: Out of the 86 tested CRKP isolates, 64 (74.4%) were carbapenemase-producing isolates. The blaOXA-48 gene was the most predominant carbapenemase gene, detected in 65.1% (n = 56) of isolates. The blaNDM gene was detected in only 9.3% (n = 8) of isolates; three were found to be co-harbored with blaVIM. Interestingly, one isolate of CRKP was found to have carbapenemase genes (blaNDM, blaVIM and blaKPC), which was associated with COVID-19 patient. CONCLUSIONS: The incidence of carbapenemase-producing K. pneumoniae in Jazan hospitals seemed to be high, confirming the continued prevalence of carbapenem resistance in Saudi Hospitals. We report K. pneumoniae strain with triple carbapenemase genes in southern Saudi Arabia. The emergence of such an isolate could threaten patients and healthcare workers and requires great attention to rapid interventions to avoid further dissemination, particularly during the COVID-19 pandemic.

Clostridioides (Clostridium) difficile: A silent nosocomial pathogen.

Clostridioides difficile (C. difficile) infection is still a threat to many healthcare settings worldwide. Clostridioides difficile epidemiology has changed over the last 20 years, largely due to the emergence of hypervirulent and antimicrobial-resistant C. difficile strains. The excessive use of antimicrobials, the absence of optimal antibiotic policies, and suboptimal infection control practices have fueled the development of this pressing health issue. The prudent use of antimicrobials, particularly broad-spectrum agents, and simple infection control measures, such as hand hygiene, can significantly reduce C. difficile infection rates. Moreover, the early detection of these infections and understanding their epidemiological behavior using accurate laboratory methods are the cornerstone to decreasing the incidence of C. difficile infection and preventing further spread. Although there is no consensus on the single best laboratory method for the diagnosis of C. difficile infection, the use of 2 or more techniques can improve diagnostic accuracy, and it is recommended.

New insights of bacterial communities in fermented vegetables from shotgun metagenomics and identification of antibiotic resistance genes and probiotic bacteria.

Consumption of fermented foods has grown worldwide due to the purported health benefits. It is thus critical to understand fermented foods microbiome that mainly influences the quality and safety of these foods. This study identified bacterial communities, including functional profiles of probiotics and antimicrobial resistance genes (ARGs), in pickled vegetables commonly consumed in the Middle Eastern, African, and Asian sub-continent regions. Eighteen samples from six pickled vegetables were collected from local markets in Saudi Arabia and analyzed using shotgun metagenomic sequencing. Statistical analyses revealed significant distance and separate clustering of bacterial communities among the different pickle types. Species of Levilactobacillus namurensis, Lentilactobacillus buchneri, Lentilactobacillus parafarraginis, Lactiplantibacillus pentosus, Pectobacterium carotovorum, Leuconostoc carnosum, Weissella confuse were found in a range of dominance in most of the samples. Binning revealed 33 high-quality, metagenome-assembled genomes (MAGs), including 4 MAGs representing putatively novel species of Lactobacillus, Alcanivorax, and Dichelobacter. Moreover, 285 ARGs and variants produce resistance against 20 classes of antibiotics were retrieved, mostly from Enterobacteriaceae contigs. The metagenomes harbored relatively high abundances of carbohydrate fermentation enzymes, as well as metabolic pathways for amino acid metabolism, cofactors and vitamins biosynthesis. Overall, by providing a comprehensive overview of bacterial communities and probiotic bacteria in pickled vegetables, the results suggest the need for more hygienic processing to avoid Enterobacteriaceae contamination and ARG spread.

Draft genome sequence of colistin-resistant and extended-spectrum ß-lactamase (ESBL)-producing multidrug-resistant Escherichia coli isolated from poultry meat.

OBJECTIVES: The presence of multidrug-resistant bacteria in the food chain represents a major public-health concern and has significant economic repercussions. Here we describe the draft genome sequence of serotype O21:H16, multilocus sequence typing (MLST) ST3270 and ribosomal MLST rST120701, multidrug-resistant Escherichia coli Ch7_4SIAU, which was isolated from poultry meat collected from a local market in Jeddah, Saudi Arabia. METHODS: Ch7_4SIAU was grown on MacConkey agar and was identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). An Illumina MiSeq platform was used to generate 2.4 million paired-end reads of 150 bp. RESULTS: The draft genome of Ch7_4SIAU is ∼5.1 Mb with a GC content of ∼50% and contains five plasmid replicons [ColpVC, IncFII, IncHI2, IncI(Gamma) and IncX1]. The isolate is resistant to third- and fourth-generation cephalosporins. It carries the mobile colistin resistance gene mcr-1.1 and the extended-spectrum ß-lactamase (ESBL) gene blaCTXM-15 as well as the astA gene encoding enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1). CONCLUSION: This report presents the first draft genome of ESBL-positive E. coli isolated from poultry meat in Saudi Arabia. The findings can be used as a reference for genomic epidemiological studies of antimicrobial resistance in Enterobacteriaceae in the local food chain.

Dissemination of VIM-producing Pseudomonas aeruginosa associated with high-risk clone ST654 in a tertiary and quaternary hospital in Makkah, Saudi Arabia.

To the best of our knowledge, no molecular surveillance that has been conducted to identify the most common clones of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in western Saudi Arabia. Therefore, this study aimed to identify genetic diversity and the most common CRPA clones in this region. Thirty-five CRPA isolates were collected from a tertiary and quaternary hospital in Makkah. bla VIM was the most common carbapenemase-encoding gene (11 CRPA isolates), while blaGES was reported in only three isolates. CRPA isolates were subjected to multi- locus sequence typing and showed relatively high genetic diversity with 20 sequence types. Approximately one-third (31.4%) of the CRPA isolates belonged to two high-risk clones (ST235 and ST654). This troublesome finding raises serious concerns about the emergence and further dissemination of CRPA high-risk clones in local hospitals and suggest that surveillance programs should be established in this region to monitor and control clonal dissemination of all multidrug resistant bacteria, including CRPA.

A simple STAR Element Repetitive-PCR (SER-PCR) typing method for the rapid characterization of Staphylococcus aureus.

INTRODUCTION: Staphylococcus aureus repeat (STAR) elements are abundant repeat sequences that are highly variable in numbers and locations within the genome of S. aureus. The evolutionary variation of these repeats occurs rapidly over time and may correlate with their evolutionary lineage. Therefore, the aim of this study is to analyze STAR elements to develop a simple PCR-based typing technique that can be used routinely in clinical laboratories. METHODOLOGY: The genomes of 10 reference strains of S. aureus were analyzed to identify the number and location of STAR elements. One pair of PCR primers was designed to target the variable region of these elements. The designed primers were first evaluated against 13 well-known reference strains of S. aureus and eventually evaluated against 40 well-characterized clinical isolates. RESULTS: STAR element repetitive-PCR (SER-PCR) showed good typeability with acceptable discriminatory power against the reference and clinical strains. The new method clearly differentiated between community-associated methicillin-resistant S. aureus (CA-MRSA) and hospital-associated (HA-MRSA) strains. Phylogenetic analysis of the SER-PCR patterns and multilocus sequence typing (MLST) data showed good correlation between the two methods. CONCLUSIONS: This study provides a simple genotyping method for S. aureus based on the variation of the amplicon size of a single set of primers and rapidly extracted DNA. The method is rapid with acceptable discriminatory power and does not require a sophisticated apparatus or special expertise. The results suggest that SER-PCR could be routinely useful in any clinical microbiology laboratory, particularly in developing countries.

Impact of mass migrations on the clonal variation of clinical Staphylococcus aureus strains isolated from the Western region of Saudi Arabia.

OBJECTIVES: A rapid molecular typing system was used to determine the impact of mass migration on the clonal variation of Staphylococcus aureus isolates recovered from King Abdulaziz University Hospital (KAUH) Jeddah, in the western region of Saudi Arabia. This region experiences an annual influx of millions of pilgrims. METHODS: SmaI-multiplex PCR typing (SMT) was used for the initial analysis of strains and the resulting data subsequently supported by Multi-Locus Sequence Typing (MLST). RESULTS: A total of 89 S. aureus isolates were SMT typed and revealed a high degree of genetic variation, with 40 SMT profiles detected among the isolates. Representatives of all forty SMT types were subsequently analysed by MLST, identifying 26 sequence types. A novel sequence type (ST), named ST3303, was identified in two methicillin-sensitive S. aureus (MSSA) isolates. MSSA strains exhibited more diversity than methicillin-resistant S. aureus (MRSA) strains, with community acquired MSSA and MRSA strains reaching alarmingly high levels. CONCLUSION: The relatively high degree of genetic diversity found among S. aureus isolates of single hospital was attributed to the fact that Jeddah is the principal gateway to Mecca, visited each year by millions of pilgrims from many countries. The observed diversity clearly reflects the impact of such mass migrations in the rapid dissemination of strains world-wide. Our findings suggest the importance of surveillance programmes in locations affected by mass migrations, both to monitor their impact on endemic strains and for the detection of pandemic strains. SMT provides a cost-effective and sensitive typing method for achieving this objective.

Routine detection of carbapenem-resistant gram-negative bacilli in clinical laboratories. A review of current challenge.

The detection of carbapenem-resistant organisms (CROs) represents a substantial challenge for many clinical laboratories. In this review, several phenotypic and non-phenotypic methods for detecting CROs are discussed. However, no consensus has yet been reached with regards to the single most optimal method. Due to differences in carbapenem-resistant activity between carbapenemases, the simultaneous use of 2 or more phenotypic detection methods can improve the detection of CROs compared with a single technique. Molecular methods are currently favored because the majority can be performed rapidly with a high level of accuracy. Whole-genome sequencing (WGS) yields unambiguous data pertaining to complete analysis of the entire genome and may ultimately become a highly powerful tool in routine clinical settings. However, WGS is still relatively expensive and requires an automated data interpretation system. The routine implementation of this technique in clinical laboratories may not occur for several years, particularly in developing countries.

The emergence of carbapenem-resistant Klebsiella pneumoniae isolates producing OXA-48 and NDM in the Southern (Asir) province, Saudi Arabia.

OBJECTIVES: To identify the prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and the most common types of cabapenemases among CRKP in the Southern (Asir) province hospitals, Saudi Arabia. Methods: The cross-sectional study was conducted between late April and September in 2015. A total of 54 Klebsiella pneumoniae (K. pneumoniae) isolates with reduced sensitivity to carbapenems were obtained from various clinical specimens of the 2 largest hospitals in the Southern province. Minimum inhibitory concentrations (MICs) of carbapenems were confirmed using E-test. Molecular detection of the most common carbapenemase genes (blaIMP, bla-carbapenem-hydrolyzing oxacillinase [OXA-48], blaVIM, bla-New Delhi metallo-ß-lactamas [NDM], and blaKPC) was performed using multiplex-polymerase chain reaction. Results: The current study found that increasing age and intensive care unit admission were associated with CRKP isolation. The major type of carbapenemases was OXA-48 with 81.5% (n=44) and it seems to reach an endemic level. New Delhi metallo-ß-lactamas (NDM) was the second most frequent carbapenemase by 7.4% (n=4) of isolates while Verona integron-encoded metallo-ß-lactamase (VIM) was reported only in one isolate. Conclusion: Saudi Arabia receives large numbers of visitors and migrant workers from OXA-48 and NDM endemic countries such as Turkey, India, and Pakistan every year.

Epidemiology of carbapenem-resistant Klebsiella pneumoniae colonization: a surveillance study at a Turkish university hospital from 2009 to 2013.

Between June 2009 and December 2013, 4105 patients were screened for carbapenem-resistant Klebsiella pneumoniae (CR-Kp) colonization in a tertiary care university hospital. The antimicrobial susceptibility and resistance determinants of 279 (6.8%) CR-Kp isolates from single patients were investigated. Additional analysis was performed to evaluate the characteristics and various risk factors for infection in patients with colonization. Of the 279 isolates, 270 harboured OXA-48-like enzymes, and a single isolate harboured IMP-type carbapenemase. A high proportion of isolates were susceptible to carbapenems - except ertapenem. All isolates were susceptible to amikacin and most (94%) were susceptible to colistin and fosfomycin. There was consistent high-level resistance for all isolates to temocillin, piperacillin-tazobactam, amoxicillin-clavulanate and ticarcillin-clavulanate. When colonized and infected patients were compared, only prior carbapenem administration (P = 0.003), was found to be significantly associated with patients with CR-Kp infection.

Composition of soil microbiome along elevation gradients in southwestern highlands of Saudi Arabia.

BACKGROUND: Saudi Arabia is mostly barren except the southwestern highlands that are susceptible to environmental changes, a hotspot for biodiversity, but poorly studied for microbial diversity and composition. In this study, 454-pyrosequencing of 16S rRNA gene hypervariable region V6 was used to analyze soil bacterial community along elevation gradients of the southwestern highlands. RESULTS: In general, lower percentage of total soil organic matter (SOM) and nitrogen were detected in the analyzed soil samples. Total 33 different phyla were identified across the samples, including dominant phyla Proteobacteria, Actinobacteria and Acidobacteria. Representative OTUs were grouped into 329 and 508 different taxa at family and genus level taxonomic classification, respectively. The identified OTUs unique to each sample were very low irrespective of the altitude. Jackknifed principal coordinates analysis (PCoA) revealed, overall differences in the bacterial community were more related to the quantity of specific OTUs than to their diversity among the studied samples. CONCLUSIONS: Bacterial diversity and soil physicochemical properties did not show consistent changes along the elevation gradients. The large number of OTUs shared between the studied samples suggest the presence of a core soil bacterial community in the southwestern highlands of Saudi Arabia.

SmaI restriction site-based multiplex PCR for typing of hospital- and community-acquired Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen, and morbidity and mortality rates associated with this pathogen have increased markedly in recent years. MRSA strains are generally resistant to several classes of antibiotics and are therefore difficult and costly to treat. A major issue is to identify the sources of MRSA infections and to monitor their epidemic spread. In this study, we report the development of a typing technique for S. aureus, based on single-nucleotide polymorphism (SNP) variations in and around SmaI-restriction sites (CCCGGG). An assessment of the SmaI restriction site-based multiplex PCR (SmaI-multiplex PCR) typing (SMT) with respect to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed a high level of concordance in the clustering of the test strains. The SmaI-multiplex PCR was found to be more discriminatory than MLST/staphylococcal cassette chromosome mec (SCCmec) typing but less discriminatory than PFGE. SMT can provide real-time information for the investigation of ongoing S. aureus hospital outbreaks. SMT meets the criteria of a practical typing method: it is simple, reproducible, and highly discriminatory and does not require expensive equipment or specialist expertise. Consequently, SmaI-multiplex PCR has the potential to be used in routine clinical microbiology laboratories.