Multiplex gyrB PCR Assay for Identification of Acinetobacter baumannii Is Validated by Whole Genome Sequence-Based Assays.

OBJECTIVE: A multiplex gyrB PCR assay has been used to diagnose Acinetobacter baumannii. However, this assay has not been validated against the gold standard DNA-DNA hybridization assay, which is a laborious method. DNA-DNA hybridization assay is now replaced by whole genome sequence (WGS)-based methods. Two such methods are a k-mer-based search of sequence reads using the Kraken 2 program and average nucleotide identity (ANI). The objective was to validate the gyrB PCR assay with WGS-based methods. SUBJECTS AND METHODS: We cultured 270 sequential A. baumannii isolates from the rectal swabs of 32 adult patients. The identity of the isolates was determined by gyrB PCR. The sequences of 269 isolates were determined by Illumina sequencing and the taxonomy was inferred by the Kraken 2 program and ANI. RESULTS: All the 269 isolates were confirmed as A. baumannii by Kraken 2 and ANI. CONCLUSION: The gyrB PCR assay is now validated for easy identification of A. baumannii in comparison with gold standard WGS-based assays.

Association of cytolethal distending toxin-II gene-positive Escherichia coli with Escherichia albertii, an emerging enteropathogen.

Cytolethal distending toxin (CDT)-producing Escherichia coli have been isolated from patients with diarrhea, sepsis and urinary tract infection. CDT of E. coli is divided into five types (CDT-I through CDT-V) based on differences in amino acid sequences and its genomic location. However, in our recent studies, a few strains of cdt-II gene-positive bacteria, initially identified as atypical E. coli, were re-identified as Escherichia albertii, an emerging enteropathogen, by extensive characterization including multilocus sequence (MLS) analysis and sugar utilization tests. This finding prompted us to investigate if bacteria previously identified as cdt-II gene-positive E. coli might be E. albertii. In the present study, we therefore re-examined the identity of 20 cdt-II gene-positive bacteria isolated from children with diarrhea, which were initially identified as atypical E. coli. By extensive sugar utilization tests, these bacteria showed a closer relatedness to E. albertii than E. coli, because they did not ferment any of the tested sugars including dulcitol, lactose, d-melibiose, l-rhamnose and d-xylose. Further, both phylogenetic analyses based on nucleotide sequences of 7 housekeeping genes (MLS analysis) and rpoB gene showed that all the cdt-II gene-positive bacteria belonged to a distinct lineage of E. albertii from those of E. coli and Shigella boydii. They were also positive by an E. albertii-specific PCR. Taken together, these data suggest that cdt-II gene-positive bacteria previously identified as E. coli are actually E. albertii. Therefore, we suggest a new definition for cdt-II gene-positive E. coli as E. albertii with the inclusion of CDT-II in E. albertii CDT.

What constitutes an Arabian Helicobacter pylori? Lessons from comparative genomics.

BACKGROUND: Helicobacter pylori, the human gastric pathogen, causes a variety of gastric diseases ranging from mild gastritis to gastric cancer. While the studies on H. pylori are dominated by those based on either East Asian or Western strains, information regarding H. pylori strains prevalent in the Middle East remains scarce. Therefore, we carried out whole-genome sequencing and comparative analysis of three H. pylori strains isolated from three native Arab, Kuwaiti patients. MATERIALS AND METHODS: H. pylori strains were sequenced using Illumina platform. The sequence reads were filtered and draft genomes were assembled and annotated. Various pathogenicity-associated regions and phages present within the genomes were identified. Phylogenetic analysis was carried out to determine the genetic relatedness of Kuwaiti strains to various lineages of H. pylori. The core genome content and virulence-related genes were analyzed to assess the pathogenic potential. RESULTS: The three genomes clustered along with HpEurope strains in the phylogenetic tree comprising various H. pylori lineages. A total of 1187 genes spread among various functional classes were identified in the core genome analysis. The three genomes possessed a complete cagPAI and also retained most of the known outer membrane proteins as well as virulence-related genes. The cagA gene in all three strains consisted of an AB-C type EPIYA motif. CONCLUSIONS: The comparative genomic analysis of Kuwaiti H. pylori strains revealed a European ancestry and a high pathogenic potential.

Isolation of Salmonella enterica serovar Kentucky strain ST 198 and its H2S-negative variant from a patient: implications for diagnosis.

H2S-producing multiresistant Salmonella enterica serovar Kentucky strain sequence type (ST) 198 and its non-H2S-producing variant were isolated from a patient. Whole-genome comparison showed a base addition in the gene encoding molybdenum cofactor biosynthesis protein C, which could affect H2S production in the variant. Lack of H2S production has implications for diagnosis of salmonella.

Real-time comparative evaluation of bioMerieux VITEK MS versus Bruker Microflex MS, two matrix-assisted laser desorption-ionization time-of-flight mass spectrometry systems, for identification of clinically significant bacteria.

BACKGROUND: Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) recently became available for the identification of bacteria in routine diagnostic laboratories. It is rapid and cost-effective and likely to replace phenotypic identification. This study was undertaken to compare two MALDI-TOF MS-based, Bruker Microflex MS (BMS) and VITEK MS (VMS) systems, for identification (ID) of clinically significant bacterial isolates. Clinically relevant broad diversity of bacterial isolates obtained during a 6-consecutive months of routine laboratory processing of clinical specimens were subjected to ID by the BMS and VMS in parallel with Vitek 2, a conventional phenotypic system (CPS). For the BMS, the isolates were tested in duplicates directly and after pretreatment. Identification was provided with accompanying scores according to manufacturers' instructions. With VMS, single deposits of the same sets of isolates were tested in duplicates directly on MALDI-plate. Results were interpreted according to the manufacturer's protocols. Discrepant results were resolved by 16S rRNA gene amplification and sequencing. RESULTS: A total of 806 pathogens comprising 507 Gram-negative bacilli (GNB), 16 Gram-negative cocci (GNC), 267 Gram-positive cocci (GPC), and 16 Gram-positive bacilli (GPB) were tested. BMS and VMS correctly identified isolates to genus and species levels (ID 97.3% and 93.2%, and 99.8% and 99.0%, respectively). Both systems as well as the CPS correctly identified the majority of the species in the family Enterobacteriaceae, Pseudomonas spp., and Acinetobacter baumannii. Turnaround time for identification by BMS and VMS was <20 min compared with 24-48 h by the CPS. CONCLUSIONS: VMS performed slightly better than BMS with GPC ID, especially the Streptococcus spp. Some S. mitis isolates were identified as S. pneumoniae by BMS. BMS and VMS were rapid and proved to be consistently accurate for producing bacterial identification in a fraction of time it takes for identification by CPS.

The challenges and successes of implementing a sustainable antimicrobial resistance surveillance programme in Nepal.

BACKGROUND: Antimicrobial resistance (AMR) is a major global public health concern and its surveillance is a fundamental tool for monitoring the development of AMR. In 1998, the Nepalese Ministry of Health (MOH) launched an Infectious Disease (ID) programme. The key components of the programme were to establish a surveillance programme for AMR and to develop awareness among physicians regarding AMR and rational drug usage in Nepal. METHODS: An AMR surveillance programme was established and implemented by the Nepalese MOH in partnership with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) from 1998 to 2003. From 2004 to 2012, the programme was integrated and maintained as a core activity of the National Public Health Laboratory (NPHL) and resulted in an increased number of participating laboratories and pathogens brought under surveillance. The main strategies were to build national capacity on isolation, identification and AMR testing of bacterial pathogens, establish laboratory networking and an External Quality Assessment (EQA) programme, promote standardised recording and reporting of results, and to ensure timely analysis and dissemination of data for advocacy and national policy adaptations. The programme was initiated by nine participating laboratories performing AMR surveillance on Vibrio cholerae, Shigella spp., Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. RESULTS: The number of participating laboratories was ultimately increased to 13 and the number of pathogens under surveillance was increased to seven (Salmonella spp. was added to the surveillance programme in 2002 and extended spectrum ß-lactamase producing Escherichia coli in 2011). From 1999 to 2012, data were available on 17,103 bacterial isolates. During the AMR programme, we observed changing trends in serovars/species for Salmonella spp., Shigella spp. and V. cholerae and changing AMR trend for all organisms. Notably, N. gonorrhoeae isolates demonstrated increasing resistance to ciprofloxacin. Additionally, the performance of the participating laboratories improved as shown by annual EQA data evaluation. CONCLUSIONS: This Nepalese AMR programme continues and serves as a model for sustainable surveillance of AMR monitoring in resource limited settings.

Enteropathogenic Providencia alcalifaciens: A Subgroup of P. alcalifaciens That Causes Diarrhea.

Despite being considered a normal flora, Providencia alcalifaciens can cause diarrhea. In a previous study, strain 2939/90, obtained from a diarrheal patient, caused invasion and actin condensation in mammalian cells, and diarrhea in a rabbit model. Four TnphoA mutants of 2939/90 produced negligible invasion and actin condensation in mammalian cells. Now, the parent strain and the mutants have been sequenced to locate TnphoA insertion sites and determine the effect on virulence. A TnphoA insertion was detected in the type three secretion system (T3SS) locus on a large plasmid and not in a T3SS locus on the chromosome. In 52 genomes of P. alcalifaciens surveyed, the chromosomal T3SS locus was present in all strains, including both P. alcalifaciens genomic clades, which we classified as group A and group B. Plasmid T3SS was present in 21 of 52 genomes, mostly in group A genomes, which included isolates from an outbreak of hemorrhagic diarrhea in dogs. The TnphoA insertion only in the plasmid T3SS locus affected the invasion phenotype, suggested that this locus is critical for causation of diarrhea. We conclude that a subgroup of P. alcalifaciens that possesses this plasmid-mediated T3SS is an enteric pathogen that can cause diarrheal disease.

High Prevalence of Novel Sequence Types in Streptococcus pneumoniae That Caused Invasive Diseases in Kuwait in 2018.

BACKGROUND: Multilocus sequence typing (MLST) is used to gain insight into the population genetics of bacteria in the form of sequence type (ST). MLST has been used to study the evolution and spread of virulent clones of Streptococcus pneumoniae in many parts of the world. Such data for S. pneumoniae are lacking for the countries of the Arabian Peninsula, including Kuwait. METHODS: We determined the STs of all 31 strains of S. pneumoniae from invasive diseases received at a reference laboratory from various health centers in Kuwait during 2018 by MLST. The relationship among the isolates was determined by phylogenetic analysis. We also determined the serotypes by Quellung reaction, and antimicrobial susceptibility by Etest, against 15 antibiotics belonging to 10 classes. RESULTS: There were 28 STs among the 31 isolates, of which 14 were new STs (45.2%) and 5 were rare STs (16.1%). Phylogenetic analysis revealed that 26 isolates (83.9%) were unrelated singletons, and the Kuwaiti isolates were related to those from neighboring countries whose information was gleaned from unpublished data available at the PubMLST website. Many of our isolates were resistant to penicillin, erythromycin, and azithromycin, and some were multidrug-resistant. Virulent serotype 8-ST53, and serotype 19A with new STs, were detected. CONCLUSIONS: Our study detected an unusually large number of novel STs, which may indicate that Kuwait provides a milieu for the evolution of novel STs. Novel STs may arise due to recombination and can result in capsular switching. This can impact the effect of vaccination programs on the burden of invasive pneumococcal disease. This first report from the Arabian Peninsula justifies the continuous monitoring of S. pneumoniae STs for the possible evolution of new virulent clones and capsular switching.

Different norovirus genotypes in patients with gastroenteritis in Kuwait.

Norovirus is a leading cause of acute gastroenteritis worldwide. The importance of this virus infection in Kuwait is not known. Eight out of 100 stool samples (8.0%) from children up to 5 years of age with gastroenteritis studied during 2006-2007 from one hospital, and 6 out of 70 stool samples (8.5%) from similar children studied from another hospital during 2010-2011 were positive for norovirus by RT-PCR. Out of these 170 samples studied from both hospitals, 10 samples were positive for norovirus when tested by ELISA. Phylogenetic tree analysis of norovirus strains showed that 50% of the norovirus strains belonged to genotype GII.4, and the predominant strain was GII.4 2006b. Other detected genotypes were GII.12, GII.b, GII.3, GII.8, and GII.7. This study highlights the importance of screening for norovirus infection in acute gastroenteritis and having a reporting system to understand better the epidemiology of norovirus infection in Kuwait.

Whole genome sequencing and comparative genomic analyses of two Vibrio cholerae O139 Bengal-specific Podoviruses to other N4-like phages reveal extensive genetic diversity.

BACKGROUND: Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I (ϕVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ϕJA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group. METHODS: Host-range analysis and plaque morphology screening were done for both ϕJA1 and ϕVchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed. RESULTS: The host specificity of ϕJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ϕVchO139-I produced a larger halo around the plaques than ϕJA1. Restriction profiles of ϕJA1 and ϕVchO139-I genomes were also different. The genomes of ϕJA1 and ϕVchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of ϕJA1 and ϕVchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family. CONCLUSIONS: The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of ϕJA1 could be demonstrated experimentally. Our data pave the way for potential use of ϕJA1 and ϕVchO139-I in Vibrio cholerae typing and control.

In vitro susceptibility of Campylobacter jejuni from Kuwait to tigecycline & other antimicrobial agents.

BACKGROUND & OBJECTIVES: There is an increasing frequency of resistance of Campylobacter jejuni to antimicrobial agents making treatment difficult. In this study, the in vitro susceptibility of C. jejuni isolates collected over an eight year period was tested against tigecycline, a glycylcycline, the previously tested antimicrobial agents in Kuwait, ciprofloxacin, erythromycin and tetracycline, and other antimicrobial agents not previously tested in Kuwait, amoxicillin-clavulanic acid, gentamicin, imipenem and meropenem. METHODS: A total of 97 C. jejuni isolates from diarrhoeal stools of Kuwaiti patients during 2002-2010 were studied for susceptibility to the above antimicrobial agents by E test. RESULTS: Erythromycin resistance increased from 5.0 per cent in 2002-2003 to 13.8 per cent in 2007-2010. The figures for ciprofloxacin resistance for the same periods were 53 and 65.5 per cent, respectively. Tetracycline resistance increased from 40.0 per cent in 2003-2006 to 62.1 per cent in 2007-2010 (P=0.05). However, all isolates were uniformly susceptible to tigecycline and other antimicrobial agents. INTERPRETATION & CONCLUSIONS: There was a progressive increase in the prevalence of resistance to ciprofloxacin, erythromycin and tetracycline. As all isolates were uniformly susceptible to tigecycline, this antimicrobial agent can be considered as a potential candidate for treatment in clinical studies.

Multidrug-Resistant and Extensively Drug-Resistant Escherichia coli in Sewage in Kuwait: Their Implications.

In Kuwait, some sewage is discharged into the sea untreated, causing a health risk. Previously, we investigated the presence of pathogenic E. coli among the 140 isolates of E. coli cultured from the raw sewage from three sites in Kuwait. The aim of the current study was to characterize the antimicrobial resistance of these isolates and the implications of resistance. Susceptibility to 15 antibiotic classes was tested. Selected genes mediating resistance to cephalosporins and carbapenems were sought. ESBL and carbapenemase production were also determined. Two virulent global clones, ST131 and ST648, were sought. A total of 136 (97.1%), 14 (10.0%), 128 (91.4%), and 2 (1.4%) isolates were cephalosporin-resistant, carbapenem-resistant, multidrug-resistant (MDR), and extensively drug-resistant (XDR), respectively. Among the cephalosporin-resistant isolates, ampC, blaTEM, blaCTX-M, blaOXA-1, and blaCMY-2 were found. Eighteen (12.9%) samples were ESBL producers. All carbapenem-resistant isolates were negative for carbapenemase genes (blaOXA-48, blaIMP, blaGES, blaVIM, blaNDM, and blaKPC), and for carbapenemase production. Resistance rates in carbapenem-resistant isolates to many other antibiotics were significantly higher than in susceptible isolates. A total of four ST131 and ST648 isolates were detected. The presence of MDR and XDR E. coli and global clones in sewage poses a threat in treating E. coli infections.

The whole-genome molecular epidemiology of sequential isolates of Acinetobacter baumannii colonizing the rectum of patients in an adult intensive care unit of a tertiary hospital.

IMPORTANCE: Acinetobacter baumannii is a multidrug-resistant nosocomial pathogen that colonizes and infects debilitated patients in the ICU. There is very little information on the genomic characteristics of colonizing strains. This information is important to understand the evolution of lineages of A. baumannii that develop resistance while patients receive antibiotic treatment in the ICU. Our study demonstrated different patterns of colonization of the rectum of ICU patients with different STs of A. baumannii while one ST colonized all patients. Some STs carried more antibiotic resistance genes compared to others. However, there was a correlation between ST and a particular resistance gene profile. Our results further elucidate the dynamics of enteric colonization of this opportunistic pathogen.

Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni.

BACKGROUND: Campylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin. RESULTS: A number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID(50)] of 1-2 µg/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50°C, and partially inactivated at 60-70°C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin. CONCLUSIONS: We report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.

Virulence and phylogenetic groups of Escherichia coli cultured from raw sewage in Kuwait.

In Kuwait, some untreated sewage is discharged into the sea which poses health risks. Therefore, we determined the virulence traits and the phylogenetic groups of E. coli cultured from raw sewage. Sewage was collected once every month for 12 months with culturing of a total of 140 E. coli isolates. E. coli was typed by the methods of Clermont. The five pathotypes of diarrheagenic E. coli (DEC), and extra-intestinal pathogenic E. coli (ExPEC) were detected by specific PCR assays. Four virulence genes which correlate with pathogenicity in animal models, were used for the first time for detection of ExPEC-vat (vacuolating auto-transporter toxin), fyuA (yersiniabactin receptor), chuA (heme-binding protein), and yfcV (major subunit of putative chaperon-usher fimbria). Most E. coli belonged to phylogenetic groups A (65[45%]) and B1 (34[24.3%]). Three (2.1%) isolates were DEC, while 14 (10%) isolates were ExPEC mostly in group B2 (57.1%). A relatively high prevalence of ExPEC in sewage has public health implications.

Cross-reactivity of outer membrane proteins of Campylobacter species with cholera toxin.

Campylobacter jejuni is a foodborne pathogen and a leading cause of diarrhoea worldwide. It is believed that a cholera toxin-like toxin (CTLT) produced by C. jejuni may mediate watery diarrhoea. However, the production of a CTLT by C. jejuni is controversial. A cholera toxin gene (ctx) homologue has not been identified in Campylobacter species. We investigated the identity of the CT cross-reactive antigen from Campylobacter species previously and the results are reviewed here. Filtrates of C. jejuni grown in four different liquid media, reported to promote CTLT production, were tested by Chinese hamster ovary (CHO) cell elongation assay for functional toxin and for reactivity with CT antibody using GM1 ganglioside ELISA (GM1 ELISA) and immunoblotting. Protein sequence of the CT antibody-reactive band was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Non-jejuni species (C. coli, C. lari, C. foetus, C. hyointestinalis and C. upsaliensis) were investigated by CHO cell assay and immunoblotting. Filtrates from seven C. jejuni reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay. However, filtrates from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains irrespective of GM1 ELISA reactivity, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein (PorA) of C. jejuni. CT antibody reacted with a C. jejuni recombinant PorA on immunoblotting. All non-C. jejuni strains were negative by CHO cell assay, but the common 53-kDa proteins reacted with CT antibody on immunoblots. The cross-reactivity of PorAs of Campylobacter species with CT may lead to the erroneous conclusion that Campylobacter species produce a functional CTLT.

The 13-valent pneumococcal conjugate vaccine (PCV13) does not appear to provide much protection on combined invasive disease due to the six PCV13 non-PCV7 serotypes 1, 3, 5, 6A, 7F, and 19A in Kuwait during 2010-2019.

Kuwait started immunizing children <2 y age with the 7-valent pneumococcal conjugate vaccine, PCV7 from August 2007. PCV7 was replaced by the 13-valent conjugate vaccine, PCV13 from August 2010. In a previous analysis of the results for the period, August 2010-July 2013 (period II), there was no evidence of serotype-specific protection for invasive disease against the additional six serotypes to PCV7 present in PCV13 (non-PCV7 serotypes) as evidenced by isolation from blood and cerebrospinal fluid in any of the age groups, <2 y, 2-5 y, 6-50 y, 51-65 y, and >65 y and all ages, compared to the pre-vaccination period, August 2003-July 2006 (period I). In the current study, we allowed additional time, August 2013-July 2019 (period III) for better vaccine effect and repeated the analysis. We did not find any significant decrease of invasive disease due to the non-PCV7 serotypes of PCV13 in period III and combined II and III periods compared to period I. However, these comparisons showed significant reductions for four of the six and total serotypes of PCV7, and total serotypes of PCV13. Reduction for total PCV13 serotypes was contributed by serotypes of PCV7. It appears that the six non-PCV7 serotypes in PCV13 do not offer much protection. Some contributory factors for the poor effect of the non-PCV7 serotypes may be related to few cases with underpowered statistical analysis, lack of vaccine coverage data, method of vaccine efficacy analysis based on vaccine serotypes relative to all serotypes and unusual rise in non-typeable isolates post vaccination that would have masked true serotypes.

Diarrhea in an infant due to Shigella flexneri 1 carrying multiple cephalosporinase-encoding genes.

BACKGROUND: Infections caused by multidrug-resistant shigellae resistant to broad-spectrum cephalosporins are becoming more prevalent in the Middle East. We report a case of severe diarrhea due to a multiresistant Shigella flexneri 1 strain carrying four different ß-lactamase genes. CASE PRESENTATION: A one-year-old Syrian infant presented with severe acute diarrhea, vomiting and dehydration. She did not respond to empirical treatment with amoxicillin-clavulanic acid followed by cefotaxime. Later, stool culture revealed S. flexneri 1 resistant to both these drugs. The patient was successfully treated with meropenem to which S. flexneri 1 was susceptible. The isolate was resistant to eight classes of antibiotics, and the whole genome sequence (WGS) identified four ß-lactamase genes (blaCTX-M-15, blaEC-8, blaOXA-1, and blaTEM-1) along with genes mediating resistance to seven other antibiotic classes. The WGS also identified several virulence genes including senA that encodes ShET-2 which induces watery diarrhea. Phylogenetically, the isolate was closely related to isolates from South Asia. CONCLUSIONS: This report highlights the emergence of extremely resistant Shigella that has acquired multiple resistance genes to cephalosporins rendering these drugs ineffective.

Antimicrobial Resistance of Serial Isolates of Acinetobacter baumannii Colonizing the Rectum of Adult Intensive Care Unit Patients in a Teaching Hospital in Kuwait.

Objectives: Outbreak and endemic isolates of Acinetobacter baumannii are known to be polyclonal. In an ongoing study, we hypothesized that the patient gut was the source of the polyclonality where genetic exchanges take place. To test the hypothesis, we collected 270 serial rectal isolates from 32 adult intensive care unit patients over 16 months and investigated their drug resistance profiles. Methods: Antimicrobial susceptibility was determined according to recommended methods. The blaIMP, blaVIM, blaSIM, blaOXA-23, blaOXA-24/40, blaOXA-51, blaOXA-48, blaKPC, blaGES, blaNDM and blaOXA-58 were sought by PCR. A subset of 42 isolates were studied for plasmid-mediated resistance. Results: Most of the 270 isolates were multidrug resistant (MDR; with resistances to meropenem of 85.18% and imipenem of 87.04%), but susceptible to colistin and trimethoprim/sulfamethoxazole. There was no correlation between the pattern of resistance and antibiotics administered to treat infections. There was no consistent pattern of resistance or content of carbapenemase genes in serial rectal isolates suggesting polyclonality of the isolates. Genes mediating production of OXA-23, OXA-24/40, IMP, and GES enzymes were carried on plasmids and they mediated resistance to all carbapenems in conjugation studies. Conclusion: A. baumannii colonizing the rectum were polyclonal, MDR, and carbapenem resistance genes were found on plasmids and some plasmids were transferable.