EPIDEMIOLOGY

MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2014.0219

Drug Resistance and Molecular Epidemiology of Aerobic Bacteria Isolated from Puerperal Infections in Bangladesh Salma Ahmed,1 Mitsuyo Kawaguchiya,2 Souvik Ghosh,2,3 Shyamal Kumar Paul,1 Noriko Urushibara,2 Chand Mahmud,1 Kamrun Nahar,4 Mohammad Akram Hossain,1 and Nobumichi Kobayashi 2

Puerperal infection is a common complication during postnatal period in developing countries. Bacterial species, drug resistance, and genetic characteristics were investigated for a total of 470 isolates from puerperal infections in Bangladesh for a 2-year period (2010–2012). The most common species was Escherichia coli (n = 98), followed by Enterococcus faecalis (n = 54), Staphylococcus haemolyticus (n = 33), Proteus mirabilis (n = 32), Staphylococcus aureus (n = 27), Klebsiella pneumoniae (n = 22), and Enterobacter cloacae (n = 21). S. aureus and Acinetobacter baumannii were isolated at a higher frequency from wound infections after cesarean section, while E. coli, E. cloacae, and K. pneumoniae were isolated from community-acquired endometritis and urinary tract infections. Resistance to third-generation cephalosporins was frequent for Enterobacteriacae, and was mainly mediated by blaCTX-M-1 group beta-lactamases. The CTX-M gene in E. coli from the four phylogroups was identified as blaCTX-M-15, and phylogroup B2 isolates with blaCTX-M-15 were classified into ST131 with O25b allele, harboring aac(6¢)-Ib-cr and various virulence factors. Carbapenemase genes blaNDM-1 and blaNDM-7 were identified in one isolate each of phylogroup A E. coli. Methicillin-resistant S. aureus isolates had type IV or V SCCmec, including isolates of ST361 (CC672), which is related to an emerging ST672 clone in the Indian subcontinent. This study revealed the recent epidemiological status of aerobic bacteria causing puerperal infections in Bangladesh, providing useful information to improve clinical practice and infection control.

cemia, but excluding obstetric pyemic and septic embolism, and septicemia during labor. Puerperal infection is a more general term than puerperal sepsis that indicates any infection after delivery (ICD-10 codes O85 and O86), including those in surgical wounds and the urinary tract, and is generically categorized as ‘‘maternal sepsis’’ by the World Health Organization (WHO) Global Burden of Disease.46 The rate of puerperal infection has been reported as 0.03–9.3 per 100 live births with case fatality rate of 4–50% that varies depending on diagnostic criteria and study settings.15 Estimated incidences of puerperal infection (per 1,000 women aged 15–49 years) are higher in Africa (6.91–7.09) and SEARO D region, including India and Bangladesh (5.1), than in other regions of the world.15 Cesarean section is reported to be associated with a higher incidence of puerperal infections than vaginal delivery according to a study in the United States.50

Introduction

I

n developing countries, maternal death is recognized as one of the major public health problems and a high level of maternal mortality ratio (MMR; number of maternal deaths per 100,000 livebirths) has been still maintained, despite the gradual decreasing trend of global MMR.26 Maternal death occurs at a higher rate in postpartum period globally (nearly 40%) than in antepartum, intrapartum, and late periods. Sepsis (puerperal sepsis), which is one of the four major causes of maternal deaths worldwide, is common in developing countries.15,43 Particularly in South Asia, the estimated number and proportion of sepsis among causes of maternal deaths is the highest in the world.43 Puerperal sepsis is an infective condition in puerperal period and is defined by ICD-10 (code O85) as a complication of the puerperium with endometritis, fever, peritonitis, and septi-

1

Department Department Department 4 Department 2 3

of of of of

Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. Hygiene, Sapporo Medical University School of Medicine, Sapporo, Japan. Biomedical Sciences, Ross University School of Veterinary Medicine, St. Kitts, West Indies. Gynecology and Obsterics, Mymensingh Medical College, Mymensingh, Bangladesh.

1

2

Puerperal infection is usually caused by bacteria that are derived from maternal vaginal flora, maternal intestinal flora, and the environment, or those related to sexually transmitted infections.31 In our previous study in Mymensingh, in centralnorthern Bangladesh, aerobic bacteria were isolated from 84% of specimens of cases with puerperal sepsis, associated with putative concomitant infection with anaerobic bacteria among half of these cases.1,2 Among the aerobic bacteria, Staphylococcus aureus was dominant (62%) and contained 46% of oxacillin-resistant strains. Other studies on the cause of puerperal infections also reported a high prevalence of S. aureus and/or enterobacteriaceae represented by Escherichia coli.4,31 However, these studies treated small sample size (50–146 specimens), and information of resistance to various antimicrobials and virulence factors of the isolated bacteria are not available. For performing efficient treatment or prevention of puerperal infections, it is indispensable to clarify microbiological causes and their characteristics. Currently, acquisition of drug resistance has been known for most bacterial species, and internationally spreading clones with peculiar resistance/virulence factors has been reported for methicillin-resistant S. aureus (MRSA) (e.g., USA300 clone), E. coli (e.g., O25b-ST131 clone), and other species,14,34,42 but their prevalence in puerperal infections has not yet been elucidated. In this study, we analyzed aerobic bacteria isolated from puerperal infections for 2 years in Bangladesh on spectrum and prevalence of bacterial species, and their susceptibility to various antimicrobials. In particular, prevalence of important resistance genes and virulence factors, and phylogenetic traits were investigated for S. aureus and E. coli, the important causes of puerperal infections.1,4 This information may provide suggestions for healthcare providers to choose appropriate antimicrobials as well as to take measures to reduce drug resistance of bacteria, resulting in promotion of infection control. Materials and Methods Collection of specimens from puerperal infections

This prospective study was conducted in the Mymensingh Medical College (MMC) hospital, in Mymensingh, Bangladesh, for patients who were diagnosed with puerperal infections for a 2 year-period between Nov. 2010 and Oct. 2012. In this study, as puerperal infections, we targeted three common diseases, that is, puerperal sepsis (endometritis), urinary tract infection, and wound infection after cesarean section. Cases of puerperal infections were selected according to the definitions proposed by WHO technical working group.45 From each patient, one of the clinical specimens, that is, endocervical swab (secretion), urine, and wound swab was obtained. Among the present study population, all the normal vaginal delivery has been done in home, while cesarean section has been performed for patients admitted to MMC hospital. Wound swab from the patients who were subjected to cesarean section was obtained at least 5 days after admission to the hospital. Therefore, wound infections were regarded as acquired in the hospital. In contrast, puerperal sepsis and urinary tract infections were considered as acquired in the community because these infections had occurred at home after normal delivery. When a patient appeared in the hospital (outpatient department or ward), a microbiologist visited the hospital by a

AHMED ET AL.

call from the specified clinical doctor, collected specimens, and simultaneously obtained clinical records of patients. The patient’s information was used to check whether the infective condition was attributable to the isolated bacteria. Since puerperal infection is usually caused by bacteria which are derived from maternal vaginal flora or intestinal flora, nonsterile specimens, that is, endocervical swabs and wound swabs were carefully collected targeting the lesion to avoid contamination, and dominant, pure culture-like colonies grown on the agar plate were regarded as a putative pathogen. Identification of bacterial species

The clinical specimens were inoculated into blood agar, MacConkey agar, nutrient agar, and mannitol salt agar plates and incubated at 37C for 24 hr aerobically. Bacterial isolates grown on the agar plates were examined by conventional microbiological methods, and their species were determined by BBL Crystal Gram-Positive ID Kit (Gram-positive cocci [GPC]) and BBL Crystal Enteric/Nonfermenter ID Kit (Gram-negative rods [GNR]) (Becton Dickinson Microbiology Systems, Cockeysville, MD). Gram-positive rods were not studied, because they are difficult to be judged as pathogens of infection. For a portion of isolates, multiplex polymerase chain reaction (PCR) was used to discriminate Enterococcus faecalis from Enterococcus faecium, and identification of E. coli, Klebsiella pneumoniae, and Proteus mirabilis, as previously described.28,37 These strains were stored in Microbank (Pro-lab Diagnostics, Richmond Hill, Canada) at - 80C until analysis. Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) against 18 antimicrobial agents based on the broth microdilution test were measured by using ‘‘Dry Plate ‘Eiken’ DP32 and DP31 (Eiken Chemical Co., Tokyo, Japan) for GPC and GNR, respectively. Breakpoints defined in the Clinical Laboratory Standards Institute (CLSI) guidelines were employed to distinguish between resistant and susceptible strains for most of the drugs examined.11 Methicillin resistance of staphylococci was defined by MICs of oxacillin ( ‡ 4 mg/ml) and cefoxitin ( ‡ 8 mg/ml), and further confirmed by PCR to detect mecA, as described next. Production of extended spectrum beta-lactamases (ESBL) was detected by doubledisk synergy test,11 using cefotaxime/clavulanic acid disk (30/10 mg/ml) and ceftazidime/clavulanic acid disk (30/ 10 mg/ml) (BD, Franklin Lakes, NJ), for isolates identified as E. coli, K. pneumoniae, Klebsiella oxytoca, and P. mirabilis. Genetic analysis of S. aureus

For S. aureus isolates, the presence of staphylococcal 16s rRNA gene, nuc, mecA, PVL gene (lukS-PV/lukF-PV), and ACME-arcA (arginine deiminase gene) was investigated by multiplex PCR assay as described by Zhang et al.51 SCCmec typing and coagulase gene (coa) typing were also performed by multiplex PCR using previously published primers and conditions.21,29 Presence of genes encoding enterotoxins, exfoliative toxins, and toxic shock syndrome toxin-1 (tst-1) were analyzed by multiplex or uniplex PCR with the use of primers as previously described.3 Sequence type (ST) was

AEROBIC BACTERIA ISOLATED FROM PUERPERAL INFECTIONS

determined according to the scheme of multilocus sequence typing (MLST).16 Bacterial clone is defined as a group of isolates that are descended from a single common ancestoral strain, having an identical genomic background with a minimal genetic difference. In this study, isolates with an identical ST were considered as belonging to a same clone, and strains with different STs belonging to a same clonal complex (CC) or ST complex were described as being clonally related. Detection and characterization of beta-lactamase genes in GNR

The presence of blaTEM, blaSHV, and blaCTX-M was confirmed by a multiplex PCR assay as previously described32 for all the isolates positive for ESBL by double-disk synergy test and other GNR showing resistance to CAZ and/or CTX. Four blaCTX-M subgroups (group 1, 2, 9, and 8/25/26) were discriminated by a multiplex PCR as previously reported,49 and a full-length nucleotide sequence of blaCTX-M was directly determined with PCR products and CTX-M subtype was analyzed by BLAST search available at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Detection and classification of plasmid-mediated AmpC gene were performed by using multiplex PCR described by Pe´rezPe´rez and Hanson.38 For GNR isolates showing resistance to imipenem and/or meropenem, detection of carbapenemase genes (blaIMP, blaSPM, blaAIM, blaVIM, blaOXA-48, blaGIM, blaBIC, blaSIM, blaNDM, blaDIM, blaKPC, blaIMI, and blaGES) was attempted by PCR using primers previously described,36,40,41 and subtype of blaNDM was determined by sequencing analysis. Genes encoding class D beta-lactamases (OXA-23-like, OXA-24-like, OXA-51-like, and OXA-58like) in Acinetobacter baumannii were detected by multiplex PCR as previously described.48 Genetic analysis of E. coli

Four main phylogenetic groups of E. coli (A, B1, B2, and D) were discriminated by triplex PCR technique and scheme described by Clermont et al.9 ST of representative isolates was determined based on Achtman scheme of MLST (http:// mlst.ucc.ie/mlst/dbs/Ecoli), using seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA).47 Recently, E. coli clone belonging to phylogenetic group B2, ST131, and O25b has been reported to have a highly pathogenic potential and spread globally. Plasmid-mediated quinolone resistance due to aac(6¢)-Ib-cr12,42 is generally associated with ESBLproducing E. coli. Deduction of O25b allele and detection of aac(6¢)-Ib-cr were performed by PCR with primers and conditions as previously described.8,10 Genes encoding virulence factors of E. coli that are mostly associated with extraintestinal infections, including adhesins, toxins, capsule, and siderophore, were detected by multiplex or uniplex PCR using primers described in several literatures.5,6,23–25,39 Statistical analysis

All the data on identified bacterial species and their frequencies in each specimen were statistically analyzed by using the SPSS version 19.0 software. A two-tailed chisquare test or Fisher’s accurate probability method (for small group sizes) was performed to examine the signifi-

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cance of difference in proportions. A p-value of < 0.05 was considered statistically significant. GenBank accession numbers

Sequences of blaNDM-7 in E coli strain PI-E402, and blaNDM-1 in E coli strain PI-E228 and A. baumanii strain PI421 determined in this study were deposited in the GenBank database under accession numbers KM485655-KM485657, respectively. Results Isolation and identification of bacterial isolates

During the 2-year study period, of 676 specimens (283 wound swab, 218 endocervical swab, and 175 urine) obtained from patients with puerperal infections, 471 isolates of aerobic bacteria (210 GPC and 261 GNR isolates) were obtained. The most common species was E. coli (n = 98), followed by E. faecalis (n = 54), Staphylococcus haemolyticus (n = 33), P. mirabilis (n = 32), S. aureus (n = 27), K. pneumoniae (n = 22), and Enterobacter cloacae (n = 21) (Table 1). Among GPC, S. aureus was isolated at a significantly higher rate from wounds (19.8%, 17/86) than endocervical swab (7.6%, 6/79) or urine (9.1%, 4/44) isolates. Similarly, A. baumannii and Burkholderia cepacia were detected mostly in wound swabs. In contrast, E. coli were isolated from endometritis and urinary tract infections (endocervical swab and urine) at a significantly higher proportion (54% and 50%, respectively) than wound infections (10%). E. cloacae was isolated most frequently from urine, while isolates of K. pneumoniae from endocervical swab were minimal. There was no significant difference in isolation rates of E. faecalis and S. haemolyticus depending on the three specimens. Polymicrobial infections were considered to be caused by S. aureus, S. haemolyticus, E. faecalis, and E. coli, among which two species were isolated from 20 specimens (11 wound, 8 endocervical swab, and 1 urine). Drug resistance of major bacterial species

Resistance patterns against various antimicrobial agents were investigated for major bacterial species (four GPC and six GNR species) (Table 2). Twenty-six percent of S. aureus were resistant to methicillin, and about 50% were resistant to ERY or LVX. More than 80% of S. haemolyticus displayed resistance to these last antibiotics. All the GPC species tested were susceptible to VCM, TEC, LZD, and FOF. GNR species exhibited high resistance rates to cephalosporins (e.g., thirdgeneration cephalosporins: CTX, 78–96%; CAZ, 61–91%). A. baumannii showed higher rates of resistance to IPM and MEM (44% and 50%, respectively) than Enterobacteriaceae ( < 9% and < 18%, respectively). All the K. pneumoniae isolates were susceptible to carbapenems. E. coli showed higher resistance rates to LVX and SXT than aminoglycosides and MIN. Except for A. baumannii, all the GNR species were nearly susceptible to FOF, while A. baumannii isolates were mostly susceptible to LVX and SXT. Genetic characterization of S. aureus

Among the 27 S. aureus isolates, seven isolates phenotypically assigned to MRSA were positive for mecA and had

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AHMED ET AL.

Table 1. Frequency (No. of Isolates) of Each Bacterial Species from Puerperal Infections Specimen (infection site) Bacterial species

No. of isolates (% in GPC/GNR)

GPC Staphylococcus aureus Staphylococcus haemolyticus Staphylococcus equorum Staphylococcus saprophyticus Staphylococcus sciuri Staphylococcus epidermidis Staphylococcus cohnii Other coagulase-negative staphylococci Enterococcus faecalis Enterococcus faecium Other Enterococcus spp. Other Gram-positive cocci Total GNR Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Proteus mirabilis Proteus vulgaris Enterobacter cloacae Other Enterobacter spp. Citrobacter spp. Pseudomonas aeruginosa Acinetobacter baumannii Burkholderia cepacia Other GNR Total

Wound (hospital)

Endocervical swab (community)

Urine (community)

27 33 11 10 8 6 6 29 54 8 4 13 209

(12.9) (15.8) (5.3) (4.8) (3.8) (2.8) (2.8) (13.8) (25.8) (3.8) (1.9) (6.2) (100)

17a 9 3 3 3 3 2 9 27 2 1 7 86

6 16 5 2 5 1 4 11 19 4 0 6 79

4 8 3 5 0 2 0 9 8 2 3 0 44

98 22 11 32 5 21 9 10 7 18 5 23 261

(35.5) (8.4) (4.2) (12.3) (1.9) (8.0) (3.4) (3.8) (2.7) (6.9) (1.9) (8.8) (100)

9b 13 4 16 4 7 2 3 5 15b 5c 7 90

47 2a 4 13 1 2 5 2 1 2 0 8 87

42 7 3 3 0 12a 2 5 1 1 0 8 84

a p < 0.05, cp < 0.01, bp < 0.001: significantly higher/lower rate among GPC or GNR isolates. GNR, Gram-negative rods; GPC, Gram-positive cocci.

type IVa- or V-SCCmec, and nine isolates were PVL gene positive (Table 3). MRSA were derived from wound (four isolates) or endocervical swab (three isolates), and classified into four coa types and four STs. Common STs of MRSA were ST361 (three isolates) and ST6 (two isolates). Only an MRSA isolate with SCCmec V (strain PI-71), belonging to coa type IVa and ST6, harbored PVL gene. Other eight PVL-positive isolates were methicillin susceptible and belonged to ST772 (CC1) or ST2884. ACME-arcA was not detected in any S. aureus isolates. Enterotoxin genes were detected in all the isolates except for an isolate having eta, while tst-1 was detected in only two MRSA isolates of ST361. Enterotoxin gene cluster (egc) seg-sei-sem-sen-seoseu was detected in S. aureus isolates belonging to CC1, CC5, and CC672. Prevalence of ESBL and carbapenemase in GNR

Among 98 E. coli isolates, 74 isolates (75.5%) harbored CTX-M gene (blaCTX-M), which was mostly assigned to group 1 (65 isolates, 87.8%) as ESBL determinant (Table 4). CIT-type AmpC genes were detected in 9 isolates, which also harbored blaCTX-M. CTX-M-positive E. coli isolates were differentiated into four phylogroups, with group A being dominant (30 isolates, 40.5%). Although sequencing was not performed, blaTEM was detected in all the GNR

examined (no. of positive isolates: E. coli, 35; K. pneumoniae, 17; K. oxytoca, 9; P. mirabilis, 20; E. cloacae, 11; A. baumannii, 2), and blaSHV was detected only in K. pneumoniae (20 isolates). O25b allele was detected by PCR in only phylogroup B2 isolates (82.6%, 19/23), and aac(6¢)-Ib-cr was found in the group B2 at the highest rate (73.9%, 17/23) than other phylogroups. It was noteworthy that 26% of CTX-Mproducing E. coli (19/74) belonged to the pandemic clone O25b-ST131.42 K. pneumoniae, K. oxytoca, and E. cloacae also harbored CTX-M-1 group gene at a high rate ( > 85.7%). Carbapenemase genes were identified for E. coli, P. mirabilis, and A. baumanii. Among E. coli, NDM gene was detected in two isolates belonging to phylogroup A, while the determinant in the remaining seven carbapenem-resistant isolates was not identified. Four P. mirabilis isolates harbored blaOXA-23. Among A. baumannii, carbapenemase genes encoding OXA23 and NDM were detected in six isolates (35.3%) and one isolate (strain PI-421) (5.9%), respectively, and the NDM gene was identified as blaNDM-1 by sequencing analysis. Genetic characterization of E. coli

Twelve representative E. coli isolates from the four phylogroups were further analyzed for their ST, resistance profiles, and virulence factors (Table 5). By sequencing analysis, CTX-M gene of all the isolates was identified as

5

31 31 32 31 31 31 31 22 0 0 32 0 0 0 0 6 27 11

(93.9) (93.9) (97.0) (93.9) (93.9) (93.9) (93.9) (66.6) (0) (0) (97.0) (0) (0) (0) (0) (18.2) (81.8) (33.3)

S. haemolyticus (n = 33) 54 54 4 54 54 54 2 54 54 26 30 54 0 0 0 0 23 54

(100) (100) (7.4) (100) (100) (100) (3.7) (100) (100) (48.1) (55.6) (100) (0) (0) (0) (0) (42.6) (100)

E. faecalis (n = 54) 8 8 6 8 8 8 6 8 8 4 7 8 0 0 0 0 8 8

(100) (100) (75.0) (100) (100) (100) (75.0) (100) (100) (50.0) (87.5) (100) (0) (0) (0) (0) (100) (100)

E. faecium (n = 8) PIP CFZ CTX CAZ FEP CPD CTM FMX ATM IPM MEM GEN AMK MIN FOF LVX SXT SAM

Antimicrobial agents 90 82 80 60 55 81 79 30 76 5 9 43 21 28 0 81 71 39

(91.8) (83.7) (81.6) (61.2) (56.1) (82.7) (80.6) (30.6) (77.6) (5.1) (9.2) (43.9) (21.4) (28.6) (0) (82.7) (72.4) (39.8)

E. coli (n = 98) 21 21 21 14 18 21 21 3 20 0 0 15 3 9 0 14 22 16

(95.5) (95.5) (95.5) (63.6) (81.8) (95.5) (95.5) (13.6) (90.9) (0) (0) (68.2) (13.6) (40.9) (0) (63.6) (100) (72.7)

K. pneumoniae (n = 22) 10 10 10 9 8 10 10 8 10 1 2 9 6 4 1 9 10 9

(90.9) (90.9) (90.9) (81.8) (72.7) (90.9) (90.9) (72.7) (90.9) (9.1) (18.2) (81.8) (54.5) (36.4) (9.1) (81.8) (90.9) (81.8)

K. oxytoca (n = 11) 19 21 19 19 17 20 19 12 19 1 1 12 4 4 0 13 14 17

(90.5) (100) (90.5) (90.5) (81.0) (95.2) (90.5) (57.1) (90.5) (4.8) (4.8) (57.1) (19.0) (19.0) (0) (61.9) (66.7) (81.0)

E. cloacae (n = 21)

3 23 25 24 22 25 23 5 16 1 3 24 11 26 0 28 25 4

(9.4) (71.9) (78.1) (75.0) (68.8) (78.1) (71.9) (15.6) (50.0) (3.1) (9.4) (75.0) (34.4) (81.3) (0) (87.5) (78.1) (12.5)

P. mirabilis (n = 32)

No. of resistant isolates (%), GNRa

18 18 16 16 12 18 18 17 17 8 9 17 8 0 3 12 14 6

(100) (100) (88.9) (88.9) (66.7) (100) (100) (94.4) (94.4) (44.4) (50.0) (94.4) (44.4) (0) (16.7) (66.7) (77.8) (33.3)

A. baumannii (n = 18)

Resistance to individual antimicrobial agent was judged according to the guidelines of Clinical Laboratory Standards Institute (CLSI)11. For antimicrobials whose resistance is not defined by CLSI guidelines, EUCAST breakpoints17 (staphylococcus spp.: FOF, > 32 mg/ml) and the following definitions (MIC) were employed to determine resistance for each species. S. aureus, S. hemolyticus: ABK, > 4 mg/ml. E. coli, K. pneumoniae, K. oxytoca, E. cloacae, P. mirabilis: CTM, FMX, > 16 mg/ml. A. baumannii: CPD, > 4 mg/ml; CFZ, CTM, FMX, ATM, > 16 mg/ml; FOF, > 128 mg/ml. Highlevel resistance to aminoglycosides for enterococcus spp. was not examined. ABK, Arbekacin; AMK, Amikacin; AMP, Ampicillin; ATM, Aztreonam; CAZ, Ceftazidime; CFZ, Cefazolin; CLI, Clindamycin; CMZ, Cefmetazole; CPD, Cefpodoxime; CTM, Cefotiam; CTX, Cefotaxime; ERY, Erythromycin; FEP, Cefepime; FMX, Flomoxef; FOF, Fosfomycin; FOX, Cefoxitin; GEN, Gentamicin; IPM, Imipenem; LVX, Levofloxacin; LZD, Linezolid; MEM, Meropenem; MIN, Minocycline; OXA, Oxacillin; PIP, Piperacillin; SAM, Ampicillin-Sulbactam; SXT, Sulfamethoxazole-Trimethoprim; TEC, Teicoplanin; VAN, Vancomycin.

a

7 7 20 7 7 7 7 0 0 0 13 1 0 0 0 2 15 4

OXA FOX AMP CFZ CMZ FMX IPM GEN ABK MIN ERY CLI VAN TEC LZD FOF LVX SXT

(25.9) (25.9) (74.1) (25.9) (25.9) (25.9) (25.9) (0) (0) (0) (48.1) (3.7) (0) (0) (0) (7.4) (55.6) (14.8)

S. aureus (n = 27)

Antimicrobial agents

No. of resistant isolates (%), GPC a

Table 2. Resistance Rates of Major Bacterial Species from Puerpeal Infections Against Antimicrobial Agents

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AHMED ET AL.

Table 3. Genotypes and Virulence Factors of Staphylococcus aureus Strains Isolated from Puerperal Infections CC

ST

coa type

mecA/ Specimen SCCmec type PVL tst-1 eta

Srtrain ID a

PI-118 , 120, 310, 311, 313 PI-158 PI-272a, 273

Wound

-

+

Urine Wound

-

+ -

PI-358

Urine

-

-

6 6 IVa 6 6 IVa 12 717 VIIa 12 12 VIIa 12 12 VIIa 12 12 VIIa 96 1930 VIa 672 361 Ia

PI-71 PI-417 PI-238 PI-281a, 131B, 297B PI-348 PI-363 PI-409 PI-476A

ES Wound ES Wound Urine Urine ES Wound

+ /V + /IVa + /IVa + /V

+ -

672 672 672

PI-459 PI-563B PI-394A PI-392 PI-420 PI-414 PI-393A PI-129

Wound Wound ES ES Wound Wound ES Wound

+ /V + /IVa + /IVa -

+ + -

1

772 IVa

1 5

772 IVa 5 IIa

5

5 IIa

361 361 672 1290 2884 2884 2885 2886

Ia Ia Ia Va IIIa IIIa VIIa VIa

+ + +

+

Enterotoxin genes sea, sec, seg, sei, sel, sem, sen, seo, seu sea, seg, sei, sem, sen, seo, seu sed, seg, sei, sem, sen, seo, seu, sej, ser sed, sei, sel, sem, sen, seo, seu, sej, ser sea sec sep sep sec, seg, sei, sep sec, sei, sep sea, sec, sei, sel sec, seg, sei, sel, sem, sen, seo, seu seg, sei, sem, sen, seo, seu sec, sei, sem, sen, seo, seu seg, sei, sem, sen, seo, seu seu seg, sei, sem, seo, sep seg, sei, sel, sep sea, sec, sei, sel

The following virulence factors were not detected: etb, etd, seb, see, seh, sek, seq, ses, set. a Strain whose ST was determined. CC, clonal complex; ES, endocervical swab; ST, sequence type.

blaCTX-M-15, and NDM genes were assigned to blaNDM-1 and blaNDM-7 for strain PI-E228 (ST90) and PI-E402 (ST167), respectively. Three phylogroup B2 isolates (PIE66, PI-E123, and PI-E201) with blaCTX-M-15 derived from different specimens were classified into ST131. Compared with most of phylogroup A, B1, and D isolates, phylogroup B2 E. coli were revealed to have evidently more virulence factors, including iha, fimH, sat, kpsM, fyuA, iutA, usp, traT, malX, hra, papC, hylA, and cnf1. Similarly, five or more virulence factors were detected in

ST410 and ST405 isolates belonging to phylogroup A and D, respectively. Discussion

This study is the first large-scale investigation on epidemiology and genetic characterization of aerobic bacteria from different types of puerperal infections. It was revealed that incidences of some causative bacteria were different depending on infection type, namely, high incidence of

Table 4. Beta-Lactamase Genes Detected in Gram-Negative Bacteria Frequently Isolated from Puerperal Infections Bacterial species (no. of isolates) E. coli (98)

K. pneumoniae (22) K. oxytoca (11) P. mirabilis (32) E. cloacae (21) A. baumannii (18)a

blaCTX-M group

blaCTX-M

blaAmpC ( + ) group 1 group 9 NI (CIT-family)

Carbapenemase gene blaOXA23

blaNDM

PCR (E. coli) O25b aac(6¢)-Ib-cr

74 Phylogroup A (30) B1 (8) B2 (23) D (13)

65

8

1

9

0

2

19

44

30 8 15 12

0 0 7 1

0 0 1 0

6 3 0 0

0 0 0 0

2 0 0 0

0 0 19 0

19 4 17 4

21 9 1 19 0

21 9 1 18 0

0 0 0 0 0

0 0 0 1 0

1 0 0 0 0

0 0 4 0 6

0 0 0 0 1

a All the A. baumannii strains possessed OXA-51 gene intrinsic to this species. NI, not identified; PCR, polymerase chain reaction.

7

410

131 38

ST38 complex

ST38 complex ST405 complex

B2

B2

D

D

131

B2

405

38

131

B2

4450 940

ST23 complex

A

90

A B1

ST23 complex

A

167

542

ST10 complex

A

ST

A

ST complex

Phylogroup

PI-E466

PI-E447

PI-E25

PI-E123

PI-E201

PI-E66

PI-E119 PI-E535

PI-E11

PI-E7

PI-E228

PI-E402

Strain ID

Wound

Wound

ES

Wound

Urine

ES

Wound Wound

ES

ES

Urine

ES

Specimen

blaCTX-M-15 blaCTX-M-15 blaCTX-M-15 blaCTX-M-15

+ + -

blaCTX-M-15 blaCTX-M-15

-

blaCTX-M-15

blaCTX-M-15

-

+

blaCTX-M-15

-

blaCTX-M-15

blaCTX-M-15, blaNDM-1

-

+

blaCTX-M-15, blaNDM-7, blaCIT

-

O25b

Beta-lactamase gene

-

-

+

+

+

+

-

-

+

-

+

aac6¢Ib-cr

PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, FMX, ATM, GEN, AMK, MIN, LVX, SXT PIP, CFZ, CTM, CTX, FEP, ATM, MIN PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, ATM, GEN, AMK, MIN, LVX, SAM

PIP, CFZ, CTM, CTX, FEP, CPD, ATM, GEN, LVF, SXT

PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, ATM, LVX, SXT

PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, FMX, ATM, IPM, MEM, LVX, SXT, SAM PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, FMX, ATM, IPM, MEM, GEN, AMK, LVX, SXT, SAM PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, ATM, LVX, SXT, SAM PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, ATM PIP, CFZ, CTM, CTX, CPD, ATM PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, FMX, AZT, LVX, SXT, SAM PIP, CFZ, CTM, CTX, CAZ, FEP, CPD, ATM, GEN, LVX

Resistance profile

fimH, kpsMTIII, kpsMTK5, fyuA, iutA, traT, malX

fimH, iha, hra, afa/draBC, sat, kpsMTII, kpsMTK5, fyuA, iutA, usp, traT, malX fimH, iha, papAH, papC, sat, kpsMTII, kpsMTK5, fyuA, iutA, usp, traT, malX fimH, iha, hra, papAH, papC, sat, cnf1, hylA, kpsMTK5, fyuA, iutA, usp, traT, malX fimH, hra, afa/draBC, sat, kpsMTII, kpsMTK5, fyuA, iutA, usp, traT, malX fimH, iutA

fimH, traT traT

fimH

fimH, afa/draBC, hra, fyuA, iutA

fimH, fyuA, traT

hra, traT

Virulence factors (adhesin, toxin, capsule, siderophore, miscellaneous)

Table 5. Genotypes, Resistance/Virulence Factors, and Resistance Profiles of Representative Escherichia coli Strains

8

S. aureus and A. baumannii in hospital-acquired infection (wound infections at surgical site of cesarean section) versus E. coli in community-acquired infections (endometritis and urinary tract infections). Although S. aureus was one of the major bacteria among GPC, E. faecalis, S. haemolyticus, and other staphylococcus species showed a higher incidence than S. aureus, providing a recent trend of puerperal infections in Bangladesh. However, causative aerobic bacteria (GPC or GNR) were not isolated from about one-third of patients in this study. As previously reported, in the same study site in Bangladesh,2 it is probable that those patients were infected with anaerobic bacteria; otherwise, some Gram-positive rods which was not regarded as pathogens might have been involved in infections. Among S. aureus strains isolated in this study, a proportion of MRSA was 26% (7/27), which was lower than those reported for hospital isolates in Bangladesh (32–63%).19 SCCmec types of the MRSA were IVa or V, which are typical to community-acquired MRSA (CA-MRSA),14 while SCCmec types of hospital-acquired MRSA (I, II, and III) were not detected. Genetically diverse traits of MRSA from puerperal infections, that is, presence of different coa-types and STs, and different toxin profiles even for strains with the same genotype, are also characteristic to CA-MRSA. Isolation of such MRSA from hospital-acquired infections (wound, four isolates) suggests that these MRSA have been brought from the community. Three among the seven MRSA isolates were typed as ST361 (CC672), a single locus variant of ST672, which has been reported as an emerging MRSA clone in India associated with type IVa or V-SCCmec and egc.27 Isolates belonging to the same CC (each of CC1, 5, 6, 12, 672) were considered to be clonally related, that is, derived from the common ancestral strain among each CC because they have identical coa type and similar enterotoxin gene profiles. This study conducted in central-northen Bangladesh revealed that GNR from puerperal infections are highly resistant to penicillin and cephalosporins. Similarly, high resistance rates to cephalosporins ( > 60%) were reported for enterobacteria from urinary tract infections in a study in eastern Bangladesh,30 suggesting widespread of beta-lactam resistance in GNR. Most of E. coli, Klebsiella spp., and E. cloacae isolates had blaCTX-M-1 group as an ESBL determinant, and the CTX-M genes of the 12 representative isolates of E. coli from the four phylogenetic groups were identified as blaCTX-M-15. Among these E. coli with CTX-M15, we detected three phylogroup B2 isolates belonging to ST131, and a phylogroup D isolate belonging to ST405, which have been recognized as international pandemic clones with increased drug resistance and virulence, frequently causing community-onset and extraintestinal infections.12,35,42 The three B2-ST131 E. coli isolates exhibited O25b allele, and had aac(6¢)-Ib-cr and various virulence factors, as previously characterized for this clone,10,42 and were considered clonally related. Although CTX-M-type ESBL-producing E. coli was detected from environmental and household water,20,44 prevalence of ST131 E. coli from humans had not yet been reported in Bangladesh. Thus, presence of B2-ST131 and D-ST405 E. coli with blaCTX-M-15 in Bangladesh was demonstrated as human pathogen for the first time in this study. In this study, resistance rate to IPM was less than 10% in GNR, and 3 isolates harboring blaNDM were detected among 16 IPM-resistant GNR isolates examined (detection rate,

AHMED ET AL.

19%). However, in a recent study with a large number of clinical isolates in Dhaka, Bangladesh, a slightly higher rate of imipenem resistance (13.6%) in GNR and a lower blaNDM-1-positive rate among IPM-resistant isolates (3.5%) were described.22 It is possible that prevalence of IPM resistance and blaNDM may vary depending on region or disease types. Despite a still low detection rate, continuous monitoring on prevalence of blaNDM among carbapenemresistant isolates from puerperal infections may be necessary. In this study, NDM-1-producing isolate PI-E228 was assigned to ST90, a clone of E. coli belonging to phylogroup A. Isolates with such a genetic background have been described in Assam, India,33 suggesting endemic distribution of this clone in the eastern part of the Indian subcontinent. NDM-7, a novel variant of NDM-1 with two amino-acid substitutions, was identified in an E. coli isolate (PI-E402, phylogroup A-ST167) in this study, first in Bangladesh. It was notable that ST167 of isolate PI-E402 is identical to that of E. coli strain COU, from which NDM-7 was first identified.13 Since the NDM-7 is considered to have improved hydrolytic efficiency of carbapenems compared with NDM1,18 more attention should be paid toward its spread among E. coli and other GNR. It was noteworthy in this study that OXA-23-like carbapenemase gene was detected in P. mirabilis, because OXA-23 is known to be often produced by A. baumannii, but detection of blaOXA-23 has been rare in enterobacteriaceae with only a report of P. mirabilis isolates in France harboring chromosomally mediated blaOXA-23.7 This study revealed the recent status of aerobic bacteria causing puerperal infections in Bangladesh. The obtained results indicated the need for continuous surveillance of GNR harboring ESBL and carbapenemase genes, and some important clones represented by ST131 E. coli and ST361 MRSA. Acknowledgment

This study was supported by a Grant-in-Aid for Scientific Research (KAKENHI) (No. 26460804) from Japan Society for Promotion of Science ( JSPS). Disclosure Statement

The authors of this article have no commercial associations that might create a conflict of interest in connection with the submitted article. References

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Address correspondence to: Nobumichi Kobayashi, MD, PhD Department of Hygiene Sapporo Medical University School of Medicine S-1 W-17, Chuo-ku Sapporo 060-8556 Japan E-mail: [email protected]