Samar S. Boswihi

Serotypes and antibiotic resistance in Group B streptococcus isolated from patients at the Maternity Hospital, Kuwait

A total of 143 group B streptococcus (GBS) isolates collected from mothers at the Maternity Hospital in Kuwait were investigated for their serotypes and antibiotic resistance, and screened by PCR for the carriage of genes for resistance to tetracycline (tetk, tetM, tetL, tetO), erythromycin (ermA, ermB, ermC, ermTR, ermM, mefA, mefE, msrA) and aminoglycosides (aph3, ant4, ant6). All isolates were serotyped using a latex agglutination test. Most of the isolates belonged to serotypes V (38.5 %), III (20.9 %), Ia (7.7 %) and II (11.2 %). Sixteen isolates (11.2 %) were nontypable. All isolates were susceptible to penicillin, ampicillin and cefotaxime (MICs 0.016-0.094 µg ml(-1)) but were resistant to trimethoprim (92.3 %), tetracycline (89.5 %), minocycline (89.5 %), high-level kanamycin (76.9 %), chloramphenicol (30.0 %), erythromycin (12.6 %), clindamycin (7.0 %), high-level streptomycin (3.5 %) and ciprofloxacin (0.7 %). The tetracycline-resistant isolates contained tetM (94.5 %), tetO (3.9 %), tetL (1.6 %) and tetK (0.8 %). The erythromycin-resistant isolates contained ermB (61.1 %), ermTR (38.9 %), ermA (5.5 %), mefA (5.5 %) and mefE (11 %). All high-level kanamycin-resistant isolates contained aph3. One of the high-level streptomycin-resistant isolates contained ant6. Partial DNA sequencing of aph3 revealed sequences with 99 % similarity to aph3 found in Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis, suggesting that the GBS isolates could have acquired aph3 from other Gram-positive cocci. The high proportion of isolates with resistance to tetracycline, high-level kanamycin and trimethoprim, and the increase in the prevalence of erythromycin resistance, represents an emerging public health concern that needs further surveillance.

High prevalence of toxic shock syndrome toxin–producing epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) strains in Kuwait hospitals

This study characterized EMRSA-15 isolates obtained from patients in Kuwait hospitals for their genotypic relatedness, antibiotic resistance and carriage of virulence genes using pulsed-field gel electrophoresis (PFGE), coagulase serotyping, SCCmec subtyping, spa typing, multilocus sequence typing and DNA microarray. The isolates were resistant to trimethoprim (75.6%), ciprofloxacin (29.7%), erythromycin and clindamycin (24.3%), tetracycline (19.0%), and gentamicin and kanamycin (21.6%). All 37 isolates belonged to sequence type (ST) 22, coagulase type XI, three PFGE types and eight subtypes, ten spa types including t223 (51.3%), t852 (13.5%), t032 (8.1%), t790 (8.1%), t3107 (5.4%) and one each of t309, t2251, t3935, t5708 and t5983. Twenty-six isolates (70.2%) carried SCCmec IVa, eight isolates carried SCCmec IV and three isolates carried SCCmec IVh. All isolates carried agr1, cap5 and egc gene cluster (seg, sei, selm, seln, selo, and selu). tst (toxic shock syndrome toxin) was detected in 23 isolates. Eight isolates (21.6%) were positive for Panton-Valentine leukocidin (PVL). Genotypic analysis revealed that 62.1% of the isolates comprising ST22-IVa-t223 (51.3%) and ST22-IVa-t309/t2251/t3935/t5708 (10.8%) were CC22-[tst1+] UK EMRSA-15/Middle Eastern variant, 21.6% were CC22-PVL+ EMRSA-15 variant and 16.2% were CC22-UK EMRSA-15/Barnim clone. These results show that the tst1 positive-ST22-IVa-t223 (Middle Eastern variant) and the CC22-PVL+ EMRSA-15 variant were the dominant EMRSA-15 variants in Kuwait hospitals.

Shifts in the Clonal Distribution of Methicillin-Resistant Staphylococcus aureus in Kuwait Hospitals: 1992-2010

Background As the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is constantly changing globally, determining the prevailing MRSA clones in a local healthcare facility is important for better management of infections. This study investigated clonal composition and distribution of MRSA isolates in Kuwait’s hospitals using a combination of molecular typing methods. Materials and Methods In total, 400 non-repeat MRSA isolates were obtained between 1992 and 2010 in 13 public hospitals and were characterized using antibiogram, SCCmec typing, spa typing, and multilocus-sequence typing. Clonal assignment and detection of virulence factors and antibiotic resistance genes were performed by DNA microarray. Results The isolates were resistant to kanamycin (74.2%), erythromycin (69.5%), tetracycline (66.7%), gentamicin (61%), ciprofloxacin, (61%), fusidic acid (53.5%), clindamycin (41.5%), high-level mupirocin resistance (5.2%) and carried aphA3, aacA-aphD, ermA, ermC, mupA, tetK, tetM, fusC and far1. Molecular typing revealed 31 different MRSA clones consisting of ST239-MRSA-III (52.2%), ST22-MRSA-IV (9.2%), ST80-MRSA-IV (7.5%), ST5-MRSA-II/IV/V/VI (6.5%), ST30-MRSA-IV (3.5%), ST241-MRSA-III (2.7%), ST6-MRSA-IV (2.2%), ST36-MRSA-II (2%) and ST772-MRSA-V (1.75%). The isolates differed in the carriage of genes for enterotoxins, Panton–Valentine leukocidin (PVL), toxic shock syndrome toxin (tst-1), arginine catabolic mobile element (ACME) and exfoliative toxins. The number of clones increased from one (ST239-III-t037) in 1992 to 30 in 2010 including ST8-IV-t008 [PVL+] [ACME+] (USA300), ST772-V (Bengal Bay clone) and ST2816 identified for the first time in Kuwait. Conclusion The study revealed that the MRSA isolates belonged to diverse clones that changed in numbers and diversity overtime. Although ST239-MRSA-III, a healthcare-associated clone remained the dominant MRSA clone overtime, the newly emerged clones consisted mostly of community-associated.

Genotypes and Virulence Genes in Group B Streptococcus Isolated in the Maternity Hospital, Kuwait

Objective: To characterize group B streptococcus (GBS) isolates obtained from patients at the Maternity Hospital in Kuwait for their genotypes and carriage of virulence genes. Materials and Methods: A total of 154 GBS isolates were obtained from July 1 to October 31, 2007, from vaginal swabs (n = 95), urine (n = 46), blood (n = 4) and miscellaneous sources (n = 9). Genotypes were obtained by pulsed-field gel electrophoresis (PFGE), following digestion with SmaI or EagI restriction enzymes. PCR was used to screen for the carriage of virulence genes including: surface protein of group B streptococcus (spb1), secreted fibrinogen-binding protein (fbsB), C5a peptidase (scpB), laminin-binding protein (lmb), α- (bca) and β-subunits of the C protein (bac), resistance to protease immunity protein (rib), and phage-associated gene (pag); regulatory protein (dltR), and toxins CAMP factor (cfb), hyaluronidase (hylB) and superoxide dismutase (sodA). Results: PFGE defined 14 genotypes differentiating isolates with the same serotypes into different genetic backgrounds. All isolates contained genes for virulence factors. However, cfb (99.4%), scpB (88.3%), lmb (88.3%), bca (57.8%), sodA (55.8%) and dltR (53.9%) were the common virulence genes. In total, 144 (90.3%) of the isolates contained 3 or more virulence genes. However, while cfb, lmb and scpB occurred in all genotypes, others occurred in some but not in all genotypes. Conclusions: GBS isolates obtained at the Maternity Hospital, Kuwait, belonged to diverse genetic backgrounds with the majority carrying multiple virulence genes.

Antibiotic Resistance Trends in Methicillin-Resistant Staphylococcus aureus Isolated in Kuwait Hospitals: 2011-2015

Objective: The aim of this study was to determine antibiotic resistance trends and carriage of staphylococcal cassette chromosome mec (SCCmec) genetic elements in methicillin-resistant Staphylococcus aureus (MRSA) isolated in Kuwait hospitals to ascertain whether they were healthcare associated (HA-MRSA) or community associated (CA-MRSA). Materials and Methods: In total, 6,922 MRSA isolates obtained from different clinical samples were tested for resistance to antibiotics, urease production, and carriage of SCCmec elements. Results: All MRSA isolates were susceptible to linezolid, vancomycin, and teicoplanin. However, some isolates were resistant to kanamycin (2,979; 43%), ciprofloxacin (2,955; 42.7%), erythromycin and clindamycin (2,935; 42.4%), fusidic acid (2,858; 41.2%), gentamicin (2,665; 38.5%), tetracycline (2,652; 38.3%), and trimethoprim (2,324; 33.5%). Whereas the prevalence of resistance to most antibiotics showed annual variations, those resistant to chloramphenicol and rifampicin increased from 2.6 and 0.1% to 9.6 and 1.6%, respectively, and high-level mupirocin resistance declined from 9.3% in 2011 to 3.6% in 2015. In total, 3,244 (53.9%) of the isolates carried SCCmec IV followed by SCCmec III (1,737; 28.8%) and SCCmec V (890; 14.8%). SCCmec I (21; 0.3%) and II (79; 0.8%) occurred sporadically. A total of 3,651 (60.7%) of the isolates belonged to the CA-MRSA genotype and 2,290 isolates (38.1%) were identified as HA-MRSA. Conclusion: This study demonstrates changes in antibiotic resistance patterns of MRSA over time and reinforces the value of surveillance in detecting such changes for the benefit of infection control and patient management.

Emerging variants of methicillin-resistant Staphylococcus aureus genotypes in Kuwait hospitals

Background Frequent changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) occurring worldwide demand regular surveillance to study their composition and distribution in healthcare facilities. We investigated the genotypic characteristics of MRSA obtained in Kuwait hospitals to better understand their clonal distribution. Materials and methods A total of 1,327 MRSA isolates obtained from clinical samples in 13 Kuwait hospitals from 1 January to 31 December 2016 were investigated using antibiogram, SCCmec typing, spa typing and DNA microarray. Results The isolates belonged to six SCCmec types with the majority belonging to type IV (658; 49.5%) and type V (355; 26.7%). Two hundred and sixty-one spa types were identified with spa types t688, t304, t860, t127, t044, t311, t002, t223, t267, t019, t3841, t005, t084, t852, and t657 constituting 51.0% (n = 677) of the isolates. Among the 1,327 MRSA isolates, 102 (7.68%) isolates were identified as novel variants of internationally recognized MRSA clones. These 102 isolates were investigated further and belonged to 14 clonal complexes (CCs) with CC361 (32; 32.3%), CC30 (15; 14.7%), CC22 (13; 12.7%) and CC1 (11, 10.7%) as the dominant CCs. Eighty-one (79.4%) of the novel isolates harbored SCCmec IV or V+fusC composite genetic elements. Four isolates (3.9%) harbored unusual combinations of ccr and mec complexes comprising of CC6-MRSA [IV+fusC+ccrC], CC97-MRSA [V/VT+fusC+ccrAB2], CC121-MRSA [V/VT+fusC+ccrB4] and CC1-MRSA-pseudoSCCmec [class B mec+fusc+ccrAB1]. Forty-six (45.1%) of these isolates were positive for PVL and 89 (87.2%) were resistant to fusidic acid mediated by fusC. Conclusions The study showed the emergence of novel variants of previously recognized MRSA genotypes with unusual genetic characteristics including high prevalence of PVL and fusidic acid resistance in Kuwait hospitals. This has added to the dynamic lists of known variations in MRSA genomes which can impose serious challenges for infection control and treatment of MRSA infections.

Molecular typing of ST239-MRSA-III from diverse geographic locations and the evolution of the SCCmec III element during its intercontinental spread

ST239-MRSA-III is probably the oldest truly pandemic MRSA strain, circulating in many countries since the 1970s. It is still frequently isolated in some parts of the world although it has been replaced by other MRSA strains in, e.g., most of Europe. Previous genotyping work (Harris et al., 2010; Castillo-Ramírez et al., 2012) suggested a split in geographically defined clades. In the present study, a collection of 184 ST239-MRSA-III isolates, mainly from countries not covered by the previous studies were characterized using two DNA microarrays (i) targeting an extensive range of typing markers, virulence and resistance genes and (ii) a SCCmec subtyping array. Thirty additional isolates underwent whole-genome sequencing (WGS) and, together with published WGS data for 215 ST239-MRSA-III isolates, were analyzed using in-silico analysis for comparison with the microarray data and with special regard to variation within SCCmec elements. This permitted the assignment of isolates and sequences to 39 different SCCmec III subtypes, and to three major and several minor clades. One clade, characterized by the integration of a transposon into nsaB and by the loss of fnbB and splE was detected among isolates from Turkey, Romania and other Eastern European countries, Russia, Pakistan, and (mainly Northern) China. Another clade, harboring sasX/sesI is widespread in South-East Asia including China/Hong Kong, and surprisingly also in Trinidad & Tobago. A third, related, but sasX/sesI-negative clade occurs not only in Latin America but also in Russia and in the Middle East from where it apparently originated and from where it also was transferred to Ireland. Minor clades exist or existed in Western Europe and Greece, in Portugal, in Australia and New Zealand as well as in the Middle East. Isolates from countries where this strain is not epidemic (such as Germany) frequently are associated with foreign travel and/or hospitalization abroad. The wide dissemination of this strain and the fact that it was able to cause a hospital-borne pandemic that lasted nearly 50 years emphasizes the need for stringent infection prevention and control and admission screening.